Mir-92 inhibitors and uses thereof

ABSTRACT

The present invention provides oligonucleotide inhibitors of miR-92 and methods of using said inhibitors for inhibiting the function and/or activity of miR-92 in a subject in need thereof. The present invention also provides methods for evaluating or monitoring the efficacy of a therapeutic for promoting wound healing and selecting a subject for treatment with a therapeutic that modulates miR-92 function and/or activity.

RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 15/878,176, filed Jan. 23, 2018, which is a continuation ofU.S. patent Ser. No. 15/001,968, which issued as U.S. Pat. No. 9,885,042on Feb. 6, 2018, and claims the benefit of U.S. Provisional ApplicationSer. No. 62/105,546, filed Jan. 20, 2015, which is incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG_050_02_US_SeqList_ST25.txt, date recorded: Apr. 25, 2018, file size161 kilobytes).

FIELD OF THE INVENTION

The present invention relates generally to modulators of miR-92 functionand/or activity, for example, oligonucleotides that are miR-92inhibitors, and biomarkers for modulators of miR-92 function and/oractivity and uses thereof.

BACKGROUND OF THE INVENTION

Diabetes and non-healing diabetic foot ulcers are the leading causes ofnon-traumatic lower extremity amputation in the US. Diabetic foot ulcersfail to heal due to an insufficient blood supply, ischemia, neuropathy,poor glucose control, infection, and other contributing factors.Treatment generally includes debridement, infection control,off-loading, and may include administration of either growth factors(e.g., platelet-derived growth factor (PDGF)) or biologic dressings.

MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAsable to negatively regulate gene expression by targeting specificmessenger RNAs (mRNAs) and inducing their degradation or translationalrepression (Ambros, Nature 431:350-355 (2004); Bartel, Cell 136:215-233(2009)). A recent study has defined mRNA degradation as the predominantmechanistic effect of miRNA on its mRNA targets (Guo et al., Nature2010; 466:835-840).

MicroRNAs have been implicated in a number of biological processesincluding regulation and maintenance of cardiac function, vascularinflammation and development of vascular pathologies (see Eva Van Rooijand Eric Olson, J. Clin. Invest. 117(9):2369-2376 (2007); Chien, Nature447:389-390 (2007); Kartha and Subramanian, J. Cardiovasc. Transl. Res.3:256-270 (2010); Urbich et al., Cardiovasc. Res. 79:581-588 (2008)).miRNAs have also been reported to be involved in the development oforganisms (Ambros, Cell 113:673-676 (2003)) and are differentiallyexpressed in numerous tissues (Xu et al., Curr. Biol. 13:790-795 (2003);Landgraf et al., Cell 129:1401-14 (2007)), in viral infection processes(Pfeffer et al., Science 304:734-736 (2004)), and associated withoncogenesis (Calin et al., Proc. Natl. Acad. Sci. USA 101:2999-3004(2004)); Calin et al., Proc. Natl. Acad. Sci. USA 99(24):15524-15529(2002)).

Accordingly, modulating the function and/or activity of microRNAs maypresent therapeutic targets in the development of effective treatmentsfor a variety of conditions. However, delivery of an antisense-basedtherapeutic targeting a miRNA can pose several challenges. The bindingaffinity and specificity to a specific miRNA, efficiency of cellularuptake, and nuclease resistance can all be factors in the delivery andactivity of an oligonucleotide-based therapeutic. For example, whenoligonucleotides are introduced into intact cells they may be attackedand degraded by nucleases leading to a loss of activity. Thus, a usefulantisense therapeutic may have good resistance to extra- andintracellular nucleases, as well as be able to penetrate the cellmembrane. Conversely, if on-target effects are undesirable in tissuesand sites other than that in which the therapeutic is administered,sensitivity to nuclease degradation may limit distal tissue exposure andactivity or limit systemic toxicity.

Thus, there is a need for stable and efficacious oligonucleotide-basedinhibitors including those for such miRNAs as, for example, miR-92.There is also a need for identification of biomarkers for miRNAmodulators, for guiding treatment decisions. The oligonucleotides of thepresent invention can have advantages in potency, efficiency ofdelivery, target specificity, stability, and/or toxicity whenadministered to a subject.

SUMMARY OF THE INVENTION

The present invention provides an oligonucleotide comprising a sequenceselected from Table 1 and Table 2. The oligonucleotide can comprise atleast one non-locked nucleotide that is 2′ O-alkyl or 2′ halo modified.In some embodiments, the oligonucleotide comprises at least one LNA thathas a 2′ to 4′ methylene bridge. In some embodiments, theoligonucleotide has a 5′ cap structure, 3′ cap structure, or 5′ and 3′cap structure. In some embodiments, the oligonucleotide comprises one ormore phosphorothioate linkages. In some embodiments, the oligonucleotideis fully phosphorothioate-linked. In yet other embodiments, theoligonucleotide comprises a pendent lipophilic group. Also providedherein is a pharmaceutical composition comprising an effective amount ofthe oligonucleotide or a pharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent. In some embodiments, thepharmaceutically-acceptable carrier comprises a colloidal dispersionsystem, macromolecular complex, nanocapsule, microsphere, bead,oil-in-water emulsion, micelle, mixed micelle, or liposome.

The present invention also provides a method of reducing or inhibitingactivity of miR-92 in a cell comprising contacting the cell with anoligonucleotide disclosed herein, such as an oligonucleotide selectedfrom Table 2. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a cardiac or muscle cell. In some embodiments,the cell is involved in wound healing. In some embodiments, the cell isa fibrocyte, fibroblast, keratinocyte or endothelial cell. In yet otherembodiments, the cell is in vitro, in vivo or ex vivo.

The present invention also provides a method of promoting angiogenesisin a subject comprising administering to the subject an oligonucleotidedisclosed herein, such as an oligonucleotide selected from Table 2. Insome embodiments, the subject suffers from ischemia, myocardialinfarction, chronic ischemic heart disease, peripheral or coronaryartery occlusion, ischemic infarction, stroke, atherosclerosis, acutecoronary syndrome, coronary artery disease, carotid artery disease,diabetes, chronic wound(s), or peripheral vascular disease (e.g.,peripheral artery disease). In some embodiments, the subject is a human.

The present invention also provides a method of promoting wound healingin a subject comprising administering to the subject a miR-92 inhibitor.In some embodiments, the miR-92 inhibitor is an oligonucleotidecomprising a sequence that is at least partially complementary tomiR-92. The oligonucleotide can comprise at least one non-lockednucleotide that is 2′ O-alkyl or 2′ halo modified. In some embodiments,the oligonucleotide comprises at least one LNA that has a 2′ to 4′methylene bridge. In some embodiments, the miR-92 inhibitor is anoligonucleotide comprising a sequence of 16 nucleotides, wherein thesequence is complementary to miR-92 and comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 6,10, 11, 13 and 16 of the sequence are LNAs. In some embodiments,position 2 from the 5′ end of the oligonucleotide comprising a sequenceof 16 nucleotides is a deoxyribonucleic acid (DNA) nucleotide that is5-methylcytosine. In some embodiments, the miR-92 inhibitor is anoligonucleotide comprising a sequence of 16 nucleotides, wherein thesequence is complementary to miR-92 and comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 3,6, 8, 10, 11, 13, 14 and 16 of the sequence are LNAs. In someembodiments, the miR-92 inhibitor is an oligonucleotide comprising asequence of 16 nucleotides, wherein the sequence is complementary tomiR-92 and comprises no more than three contiguous LNAs, wherein fromthe 5′ end to the 3′ end, positions 1, 5, 6, 8, 10, 11, 13, 15 and 16 ofthe sequence are LNAs. In some embodiments, the miR-92 inhibitor is anoligonucleotide comprising a sequence of 16 nucleotides, wherein thesequence is complementary to miR-92 and comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 3,6, 9, 10, 11, 13, 14 and 16 of the sequence are LNAs. In someembodiments, the oligonucleotide has a 5′ cap structure, 3′ capstructure, or 5′ and 3′ cap structure. In some embodiments, theoligonucleotide comprises one or more phosphorothioate linkages. In someembodiments, the oligonucleotide is fully phosphorothioate-linked. Inyet other embodiments, the oligonucleotide comprises a pendentlipophilic group. In some embodiments, the miR-92 inhibitor is anoligonucleotide disclosed herein, such as an oligonucleotide selectedfrom Tables 1 or 2. In some embodiments, the subject suffers fromdiabetes, wounds, peripheral or coronary artery occlusion, or peripheralvascular disease (e.g., peripheral artery disease). In some embodiments,the subject is a human. In some embodiments, the wound is a chronicwound, diabetic foot ulcer, venous stasis leg ulcer or pressure sore. Insome embodiments, administration of the miR-92 oligonucleotide inhibitorproduces an improvement in re-epithelialization, granulation and/orneoangiogenesis of a wound in the subject during wound healing. In someembodiments, the improvement in re-epithelialization, granulation and/orneoangiogenesis (neovascularization) of the wound is as compared to thesubject receiving no treatment. In some embodiments, the improvement inre-epithelialization, granulation and/or neoangiogenesis of the wound isas compared to the subject receiving treatment with an agent known topromote wound healing. In some embodiments, the agents known to promotewound healing are growth factors. In some embodiments, the growthfactors are platelet derived growth factor (PDGF) or vascularendothelial growth factor (VEGF).

The present invention also provides an oligonucleotide comprising asequence of 16 nucleotides, wherein the sequence is complementary tomiR-92 and comprises no more than three contiguous LNAs, wherein fromthe 5′ end to the 3′ end, positions 1, 6, 10, 11, 13 and 16 of thesequence are LNAs, and wherein position 2 from the 5′ end comprises adeoxyribonucleic acid (DNA) nucleotide that is 5-methylcytosine. In someembodiments, the oligonucleotide comprises LNAs at positions 1, 3, 6, 8,10, 11, 13, 14 and 16 from the 5′ end to the 3′ end. In otherembodiments, the oligonucleotide comprises LNAs at positions 1, 5, 6, 8,10, 11, 13, 15 and 16 from the 5′ end to the 3′ end. In yet otherembodiments, the oligonucleotide comprises LNAs at positions 1, 3, 6, 9,10, 11, 13, 14 and 16 from the 5′ end to the 3′ end. The oligonucleotidecan further comprise at least one non-locked nucleotide that is2′-deoxy, 2′ O-alkyl or 2′ halo modified. In some embodiments, allnon-locked nucleotides of the oligonucleotide are 2′-deoxy modified. Insome embodiments, the oligonucleotide comprises at least one LNA thathas a 2′ to 4′ methylene bridge. In some embodiments, theoligonucleotide has a 5′ cap structure, 3′ cap structure, or 5′ and 3′cap structure. In some embodiments, the oligonucleotide comprises one ormore phosphorothioate linkages. In some embodiments, the oligonucleotideis fully phosphorothioate-linked. In yet other embodiments, theoligonucleotide comprises a pendent lipophilic group. In someembodiments, the presence of the 5-methylcyotsine at position 2 from the5′ end of the oligonucleotide inhibitor comprising a sequence of 16nucleotides confers increased in vivo, ex vivo and/or in vitro efficacyas compared to an oligonucleotide inhibitor containing the same sequenceas well as number and positions of LNAs but lacks the 5-methylcytosine.In some embodiments, the increased efficacy is evidenced by eliminationor an enhanced reduction in function and/or activity of miR-92.

The present invention also provides a method of reducing or inhibitingactivity or function of a miRNA in a cell comprising contacting the cellwith the oligonucleotide described herein comprising a sequence of 16nucleotides, wherein the sequence is complementary to a miRNA andcomprises no more than three contiguous LNAs, wherein from the 5′ end tothe 3′ end, positions 1, 6, 10, 11, 13 and 16 of the sequence are LNAs.In some embodiments, the oligonucleotide comprises LNAs at positions 1,3, 6, 8, 10, 11, 13, 14 and 16 from the 5′ end to the 3′ end. In otherembodiments, the oligonucleotide comprises LNAs at positions 1, 5, 6, 8,10, 11, 13, 15 and 16 from the 5′ end to the 3′ end. In yet otherembodiments, the oligonucleotide comprises LNAs at positions 1, 3, 6, 9,10, 11, 13, 14 and 16 from the 5′ end to the 3′ end. In someembodiments, the sequence is complementary to miR-92. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a cardiac or muscle cell. In some embodiments, the cell is involvedin wound healing. In some embodiments, the cell is a fibrocyte,fibroblast, keratinocyte or endothelial cell. In yet other embodiments,the cell is in vitro, in vivo or ex vivo.

The present invention also provides a method of promoting angiogenesisin a subject comprising administering to the subject the oligonucleotidedescribed herein comprising a sequence of 16 nucleotides, wherein thesequence is complementary to miR-92 and comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 6,10, 11, 13 and 16 of the sequence are LNAs. In some embodiments, theoligonucleotide comprises LNAs at positions 1, 3, 6, 8, 10, 11, 13, 14and 16 from the 5′ end to the 3′ end. In other embodiments, theoligonucleotide comprises LNAs at positions 1, 5, 6, 8, 10, 11, 13, 15and 16 from the 5′ end to the 3′ end. In yet other embodiments, theoligonucleotide comprises LNAs at positions 1, 3, 6, 9, 10, 11, 13, 14and 16 from the 5′ end to the 3′ end. In some embodiments, the subjectsuffers from ischemia, myocardial infarction, chronic ischemic heartdisease, peripheral or coronary artery occlusion, ischemic infarction,stroke, atherosclerosis, acute coronary syndrome, coronary arterydisease, carotid artery disease, diabetes, chronic wound(s), orperipheral vascular disease (e.g., peripheral artery disease). In someembodiments, the subject is a human.

The present invention also provides a method of treating ischemia,myocardial infarction, chronic ischemic heart disease, peripheral orcoronary artery occlusion, ischemic infarction, stroke, atherosclerosis,acute coronary syndrome, coronary artery disease, carotid arterydisease, diabetes, chronic wound(s) or peripheral artery disease in asubject comprising administering to the subject the oligonucleotidedescribed herein comprising a sequence of 16 nucleotides, wherein thesequence is complementary to miR-92 and comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 6,10, 11, 13 and 16 of the sequence are LNAs. In some embodiments, theoligonucleotide comprises LNAs at positions 1, 3, 6, 8, 10, 11, 13, 14and 16 from the 5′ end to the 3′ end. In other embodiments, theoligonucleotide comprises LNAs at positions 1, 5, 6, 8, 10, 11, 13, 15and 16 from the 5′ end to the 3′ end. In yet other embodiments, theoligonucleotide comprises LNAs at positions 1, 3, 6, 9, 10, 11, 13, 14and 16 from the 5′ end to the 3′ end. In some embodiments, the subjectis a human.

The present invention is also based, in part, on the discovery of genessignificantly regulated by miR-92. Accordingly, another aspect of thepresent invention is a method for evaluating or monitoring the efficacyof a therapeutic for modulating angiogenesis and/or treating chronicwounds in a subject receiving the therapeutic comprising: measuring theexpression of one or more genes listed in Table 3 in a sample from thesubject; and comparing the expression of the one or more genes to apre-determined reference level or level of the one or more genes in acontrol sample, wherein the comparison is indicative of the efficacy ofthe therapeutic. Another aspect of the present invention is a method forselecting a subject for treatment with a therapeutic that modulatesmiR-92 function and/or activity comprising: measuring the expression ofone or more genes listed in Table 3 in a sample from the subject,wherein the subject is treated with the therapeutic; and comparing theexpression of the one or more genes to a pre-determined reference levelor level of the one or more genes in a control sample, wherein thecomparison is indicative of whether the subject should be selected fortreatment (e.g. further treatment or continued treatment) with thetherapeutic. In some embodiments, the methods comprise a subject thatsuffers from ischemia, myocardial infarction, chronic ischemic heartdisease, peripheral or coronary artery occlusion, ischemic infarction,stroke, atherosclerosis, acute coronary syndrome, coronary arterydisease, carotid artery disease, diabetes, chronic wound(s) orperipheral vascular disease (e.g., peripheral artery disease). In someembodiments, the subject has a chronic wound, diabetic foot ulcer,venous stasis leg ulcer or pressure sore. In some embodiments, thesubject is a human.

In some embodiments, the methods further comprise performing a walk timetest on the subject, determining an ankle-bronchial index (ABI) for thesubject, performing an arteriography or angiography on the subject, orperforming a SPECT analysis on the subject. In some embodiments, thetherapeutic modulates miR-92 function and/or activity. The therapeuticcan be a miR-92 antagonist, such as a miR-92 inhibitor selected fromTables 1 and 2. In other embodiments, the therapeutic is a miR-92agonist, such as a miR-92 mimic. In some embodiments, the methodscomprise a subject that suffers from ischemia, myocardial infarction,chronic ischemic heart disease, peripheral or coronary artery occlusion,ischemic infarction, stroke, atherosclerosis, acute coronary syndrome,coronary artery disease, carotid artery disease, diabetes, chronicwound(s) or peripheral vascular disease (e.g., peripheral arterydisease). In some embodiments, the subject is a human.

Also provided herein is a method for evaluating an agent's ability topromote angiogenesis comprising: measuring the expression of one or moregenes listed in Table 3 in a cell contacted with the agent; andcomparing the expression of the one or more genes to a pre-determinedreference level or level of the one or more genes in a control sample,wherein the comparison is indicative of the agent's ability to promoteangiogenesis. In some embodiments, the method further comprisesdetermining miR-92 function, and/or activity in the cell contacted withthe agent. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a cardiac or muscle cell. In some embodiments,the cell is involved in wound healing. In some embodiments, the cell isa fibrocyte, fibroblast, keratinocyte or endothelial cell. In yet otherembodiments, the cell is in vitro, in vivo or ex vivo.

Another aspect of the present invention is a method for evaluating ormonitoring the efficacy of a therapeutic for promoting wound healing ina subject receiving the therapeutic comprising: measuring the expressionof one or more genes listed in Table 3 in a sample from the subject; andcomparing the expression of the one or more genes to a pre-determinedreference level or level of the one or more genes in a control sample,wherein the comparison is indicative of the efficacy of the therapeutic.In some embodiments, the therapeutic modulates miR-92 function and/oractivity. The therapeutic can be a miR-92 antagonist, such as a miR-92inhibitor selected from Tables 1 and 2. In other embodiments, thetherapeutic is a miR-92 agonist, such as a miR-92 mimic. In someembodiments, the methods comprise a subject that suffers from ischemia,myocardial infarction, chronic ischemic heart disease, peripheral orcoronary artery occlusion, ischemic infarction, stroke, atherosclerosis,acute coronary syndrome, coronary artery disease, carotid arterydisease, diabetes, chronic wound(s) or peripheral vascular disease(e.g., peripheral artery disease). In some embodiments, the subject is ahuman.

Also provided herein is a method for evaluating an agent's ability topromote wound healing comprising: measuring the expression of one ormore genes listed in Table 3 in a cell contacted with the agent; andcomparing the expression of the one or more genes to a pre-determinedreference level or level of the one or more genes in a control sample,wherein the comparison is indicative of the agent's ability to promotewound healing. In some embodiments, the method further comprisesdetermining miR-92 function and/or activity in the cell contacted withthe agent. In some embodiments, the cell is a mammalian cell. In yetother embodiments, the cell is in vitro, in vivo or ex vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates multiple genes significantly regulated by miR-92amodulation in human umbilical vein endothelial cells (HUVECs).

FIG. 2A-C illustrates integrin α5 expression regulation by miR-92a inHUVECs. Integrin α5 (ITGA5) transcript (FIG. 2A-B) and protein (FIG. 2C)levels are increased in response to miR-92a inhibition and decreased inresponse to miR-92a mimic. FIGS. 2A-B show the indicated concentrationsthat the indicated oligonucleotides were lipid-transfected or passivelydelivered to HUVECs, respectively. FIG. 2C shows the integrin α5 proteinlevels following lipid-mediated transfection. Passive delivery refers tounassisted oligonucleotide uptake. A (SEQ ID NO. 7), B (SEQ ID NO. 61)and C (SEQ ID NO. 62) are LNA/DNA-containing miR-92a inhibitors.“AntagomiR” is an O-methylated, cholesterol-conjugated inhibitor. D is asingle-stranded LNA/DNA control inhibitor.

FIG. 3 illustrates regulation by real time PCR of targets identified bymicroarray profiling. Four genes identified by microarray profiling areincreased in response to miR-92a inhibition and decreased in response tomiR-92a mimic, in an independent HUVEC lipid-transfection experiment.The radar plot indicates the relative expression of MAN2A1, CNEP1R1,ERGIC2, and CD93 in response to miR-92a inhibitor or mimic, normalizedto HUVECs transfected with lipid without oligonucleotide. The black lineindicates where the gene expression would be if there were no change,the red line indicates the gene expression in response to D (controloligo) transfection.

FIG. 4 illustrates a dual luciferase assay for testing of inhibitordesign activity. MiR-92a inhibitors were ranked based on their abilityto de-repress the expression of luciferase from a dual-luciferasereporter plasmid. Shown is an example set of data from the first ofthree replicate experiments.

FIG. 5A-F illustrates results of a first study examining the activity ofoligonucleotide inhibitors of miR-92 in an in vivo model of impairedwound healing. FIG. 5A illustrates the percent re-epithelialization ofwounds in the in vivo model of impaired wound healing from phosphatebuffered saline (PBS; vehicle-control), vascular endothelial growthfactor (VEGF; positive control) and oligonucleotide inhibitors of miR-92(A (SEQ ID NO 7); C (SEQ ID NO. 62)) treatment groups. FIG. 5Billustrates the percent granulation tissue ingrowth or filled in woundsin the in vivo model of impaired wound healing from PBS(vehicle-control), VEGF (positive control) and oligonucleotideinhibitors of miR-92 (A; C) treatment groups. FIG. 5C illustrates thegranulation tissue area in wounds in the in vivo model of impaired woundhealing from PBS (vehicle-control), VEGF (positive control) andoligonucleotide inhibitors of miR-92 (A; C) treatment groups. FIG. 5Dillustrates the average granulation tissue thickness across wounds(wound area divided by wound width) in the in vivo model of impairedwound healing from PBS (vehicle-control), VEGF (positive control) andoligonucleotide inhibitors of miR-92 (A; C) treatment groups. FIGS. 5E-Fillustrate the number of CD31+ endothelial cells (FIG. 5E) and thetissue area that was CD31+ (FIG. 5F)in wounds in the in vivo model ofimpaired wound healing from PBS (vehicle-control) and oligonucleotideinhibitor of miR-92 (A) treatment groups using immunohistochemistry, asan indicator of neovascularization/angiogenesis

FIG. 6A-F illustrates results of a second study examining the activityof oligonucleotide inhibitors of miR-92in an in vivo model of impairedwound healing. FIG. 6A illustrates the percent re-epithelialization ofwounds in the in vivo model of impaired wound healing from PBS(vehicle-control), VEGF (positive control), platelet derived growthfactor (PDGF; positive control) and oligonucleotide inhibitors of miR-92(A; C) treatment groups. FIG. 6B illustrates the percent granulationtissue ingrowth or filled in wounds in the in vivo model of impairedwound healing from PBS (vehicle-control), VEGF (positive control), PDGF(positive control) and oligonucleotide inhibitors of miR-92 (A; C)treatment groups. FIG. 6C illustrates the granulation tissue area inwounds in the in vivo model of impaired wound healing from PBS(vehicle-control), VEGF (positive control), PDGF (positive control) andoligonucleotide inhibitors of miR-92 (A; C) treatment groups. FIG. 6Dillustrates the average granulation tissue thickness across wounds(wound area divided by wound width) in the in vivo model of impairedwound healing from PBS (vehicle-control), VEGF (positive control), PDGF(positive control) and oligonucleotide inhibitors of miR-92 (A; C)treatment groups. FIGS. 6E-F illustrate the number of CD31+ endothelialcells (FIG. 6E) and the tissue area that was CD31+ (FIG. 6F) in woundsin the in vivo model of impaired wound healing from PBS(vehicle-control), VEGF (positive control), PDGF (positive control) andoligonucleotide inhibitor of miR-92 (A) treatment groups usingimmunohistochemistry, as an indicator of neovascularization/angiogenesis

FIG. 7 illustrates de-repression of selected miR-92a target genes byoligonucleotide inhibitors of miR-92a from one in vivo study in db/dbmouse excisional wounds as assessed by quantitative RT-PCR.

FIG. 8A-D illustrates the effects of saline (vehicle-control; FIG. 8A),PDGF (positive control; FIG. 8B), and oligonucleotide inhibitor ofmiR-92 (A; FIGS. 8C-D) treatments on the protein expression of themiR-92 target ITGA5 in wounds in the in vivo model of impaired woundhealing as evaluated using immunohistochemistry.

FIG. 9 illustrates a dual luciferase assay for testing of the effect ofthe presence of 5-methylcyostine on inhibitor design activity. MiR-92ainhibitors with or without 5-methylcytosine were analyzed based on theirability to de-repress the expression of luciferase from adual-luciferase reporter plasmid. Shown is an example set of data.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligonucleotide inhibitors that inhibitthe activity or function of miR-92 and compositions and uses thereof.Also provided herein are miR-92 agonists, such as a miR-92 mimic.

MiR-92 is located in the miR-17-92 cluster, which consists of miR-17-5p,miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92-1 (Venturiniet al., Blood 109 10:4399-4405 (2007)). The pre-miRNA sequence formiR-92 is processed into a mature sequence (3p) and a star (i.e. minoror 5p) sequence. The star sequence is processed from the other arm ofthe stem loop structure. The mature and star miRNA sequences for human,mouse, and rat miR-92 are provided:

Human mature miR-92 (i.e. hsa-miR-92a-3p) (SEQ ID NO: 1)5′-UAUUGCACUUGUCCCGGCCUGU-3′ Human miR-92a-1* (i.e. hsa-miR-92a-1-5p)(SEQ ID NO: 2) 5′-AGGUUGGGAUCGGUUGCAAUGCU-3′Human miR-92a-2* (i.e. hsa-miR-92a-2-5p) (SEQ ID NO: 3)5′-GGGUGGGGAUUUGUUGCAUUAC-3′ Mouse mature miR-92 (i.e. mmu-miR-92a-3p)(SEQ ID NO: 4) 5′-UAUUGCACUUGUCCCGGCCUG-3′Mouse miR-92a-1* (i.e. mmu-miR-92a-1-5p) (SEQ ID NO: 5)5′-AGGUUGGGAUUUGUCGCAAUGCU-3′ Mouse miR-92a-2* (i.e. mmu-miR-92a-2-5p)(SEQ ID NO: 6) 5′-AGGUGGGGAUUGGUGGCAUUAC-3′Rat mature miR-92 (i.e. rno-miR-92a-3p) (SEQ ID NO: 4)5′-UAUUGCACUUGUCCCGGCCUG-3′ Rat miR-92a-1* (i.e. rno-miR-92a-1-5p)(SEQ ID NO: 5) 5′-AGGUUGGGAUUUGUCGCAAUGCU-3′Rat miR-92a-2* (i.e. rno-miR-92a-2-5p) (SEQ ID NO: 6)5′-AGGUGGGGAUUAGUGCCAUUAC-3′

The above sequences can be either ribonucleic acid sequences ordeoxyribonucleic acid sequences or a combination of the two (i.e. anucleic acid comprising both ribonucleotides and deoxyribonucleotides).It is understood that a nucleic acid comprising any one of the sequencesdescribed herein will have a thymidine base in place of the uridine basefor DNA sequences and a uridine base in place of a thymidine base forRNA sequences.

In some embodiments, the oligonucleotide comprising a sequencecomplementary to miR-92 is a miR-92 inhibitor. The oligonucleotidecomprising a sequence complementary to miR-92 can be an oligonucleotideinhibitor. In the context of the present invention, the term“oligonucleotide inhibitor”, “antimiR”, “antagonist”, “antisenseoligonucleotide or ASO”, “oligomer”, “anti-microRNA oligonucleotide orAMO”, or “mixmer” is used broadly and encompasses an oligomer comprisingribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides or a combination thereof, that inhibitsthe activity or function of the target microRNA (miRNA) by fully orpartially hybridizing to the miRNA thereby repressing the function oractivity of the target miRNA.

The term “miR-92” as used herein includes pri-miR-92, pre-miR-92,miR-92, miR-92a, miR-92b, miR-92a-3p, and hsa-miR-92a-3p.

In some embodiments, certain oligonucleotide inhibitors of the presentinvention may show a greater inhibition of the activity or function ofmiR-92 in cells as compared to other miR-92 inhibitors. In someembodiments, the cell is a cardiac or muscle cell. In some embodiments,the cell is involved in wound healing. In some embodiments, the cell isa fibrocyte, fibroblast, keratinocyte or endothelial cell. In yet otherembodiments, the cell is in vivo or ex vivo. In some embodiments, theoligonucleotide inhibitors of miR-92 of the present invention showhigher efficacy as compared to other oligonucleotide inhibitors ofmiR-92 as measured by the amount of de-repression of a miR-92 targetsuch as a gene selected from Table 3.

The term “other miR-92 inhibitors” includes nucleic acid inhibitors suchas antisense oligonucleotides, antimiRs, antagomiRs, mixmers, gapmers,aptamers, ribozymes, small interfering RNAs, or small hairpin RNAs;antibodies or antigen binding fragments thereof; and/or drugs, whichinhibit the expression or activity of miR-92. It is possible that aparticular oligonucleotide inhibitor of the present invention may show agreater inhibition of miR-92 in cells (e.g., muscle cells, cardiaccells, endothelial cells, fiborcytes, fibroblasts, or keratinocytes)compared to other oligonucleotide inhibitors of the present invention.The term “greater” as used herein refers to quantitatively more orstatistically significantly more.

The activity of the oligonucleotide in modulating the function and/oractivity of miR-92 may be determined in vitro, ex vivo and/or in vivo.For example, when inhibition of miR-92 activity is determined in vitro,the activity may be determined using a dual luciferase assay. The dualluciferase assay can be any dual luciferase assay known in the art. Thedual luciferase assay can be a commercially available dual luciferaseassay. The dual luciferase assay, as exemplified by the commerciallyavailable product PsiCHECK™ (Promega), can involve placement of the miRrecognition site in the 3′ UTR of a gene for a detectable protein (e.g.,renilla luciferase). The construct can be co-expressed with miR-92, suchthat inhibitor activity can be determined by change in signal. A secondgene encoding a detectable protein (e.g., firefly luciferase) can beincluded on the same plasmid, and the ratio of signals determined as anindication of the antimiR-92 activity of a candidate oligonucleotide. Insome embodiments, the oligonucleotide significantly inhibits suchactivity, as determined in the dual luciferase activity, at aconcentration of about 50 nM or less, or in other embodiments, 40 nM orless, 20 nM or less, or 10 nM or less. For example, the oligonucleotidemay have an IC50 for inhibition of miR-92 activity of about 50 nM orless, 40 nM or less, 30 nM or less, or 20 nM or less, as determined inthe dual luciferase assay.

Alternatively, or in addition, the in vivo efficacy of theoligonucleotide inhibitor of a miRNA as provided herein (e.g., miR-92)may also be determined in a suitable animal model. The animal model canbe a rodent model (e.g., mouse or rat model). The oligonucleotide mayexhibit at least 50% miR-92 target de-repression at a dose of 50 mg/kgor less, 25 mg/kg or less, 10 mg/kg or less or 5 mg/kg or less. In suchembodiments, the oligonucleotide may be dosed, delivered or administeredto mice intravenously or subcutaneously or delivered locally such aslocal injection into muscle or a wound (e.g., to the wound margin orwound bed), and the oligonucleotide may be formulated in saline. In someembodiments, the application may be dosed to mice topically orintradermally (i.e., intradermal injection), such as to a wound (e.g.,to the wound margin or wound bed). The oligonucleotide inhibitor ofmiR-92 as provided herein can have increased in vivo efficacy in aparticular tissue as compared to other oligonucleotide inhibitors ofmiR-92.

In some embodiments, the in vivo efficacy of the oligonucleotide isdetermined in a suitable mouse or rat model for diabetes. In oneembodiment, the mouse model is a genetically type II diabetic mice suchas db/db mice (Jackson Cat #000642 BKS.Cg Dock(Hom) 7m+/+ Leprdb/j). Inone embodiment, the model uses full thickness cutaneous excisional punchbiopsy. In other embodiments, the model utilizes an incision, scald orburn. In such embodiments, the oligonucleotide may be dosed to miceintravenously or subcutaneously, or delivered locally such as localinjection or topical application to a wound (e.g., the wound margin orwound bed).

In these or other embodiments, the oligonucleotides of the presentinvention can be stable after administration, being detectable in thecirculation and/or target organ for at least three weeks, at least fourweeks, at least five weeks, or at least six weeks, or more, followingadministration. Thus, the oligonucleotide inhibitors of a miRNA (e.g.,miR-92) provided herein may provide for less frequent administration,lower doses, and/or longer duration of therapeutic effect as compared toother oligonucleotide inhibitors of the miRNA (e.g., miR-92).

The nucleotide sequence of the oligonucleotide can be substantiallycomplementary to a nucleotide sequence of an RNA, such as a mRNA ormiRNA. The nucleotide sequence of the oligonucleotide can be fullycomplementary to a nucleotide sequence of an RNA, such as a mRNA ormiRNA. In some embodiments, the miRNA is miR-92 or miR-92a. Theoligonucleotide comprises at least one LNA, such as at least two, atleast three, at least five, at least seven or at least nine LNAs. Insome embodiments, the oligonucleotide comprises a mix of LNA andnon-locked nucleotides. For example, the oligonucleotide may contain atleast five or at least seven or at least nine locked nucleotides, and atleast one non-locked nucleotide.

Generally, the length of the oligonucleotide and number and position oflocked nucleotides can be such that the oligonucleotide reduces miR-92function and/or activity. In some embodiments, the length of theoligonucleotide and number and position of locked nucleotides is suchthat the oligonucleotide reduces miR-92 function and/or activity at anoligonucleotide concentration of about 50 nM or less in the in vitroluciferase assay, or at a dose of about 50 mg/kg or less, or about 25mg/kg or less in a suitable mouse or rat model, each as described. Insome embodiments, the length of the oligonucleotide and number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-92 activity as determined by target de-repression, at a dose ofabout 50 mg/kg or less, or about 25 mg/kg or less in a suitable mouse orrat model, such as described herein.

The oligonucleotide of the present invention can comprise a sequence ofnucleotides in which the sequence comprises at least five LNAs, a LNA atthe 5′ end of the sequence, a LNA at the 3′ end of the sequence, or anycombination thereof. In one embodiment, the oligonucleotide comprises asequence of nucleotides in which the sequence comprises at least fiveLNAs, a LNA at the 5′ end of the sequence, a LNA at the 3′ end of thesequence, or any combination thereof, wherein three or fewer of thenucleotides are contiguous LNAs. For example, the oligonucleotidecomprises no more than three contiguous LNAs. For example, theoligonucleotide may comprise a sequence with at least five LNAs, a LNAat the 5′ end, a LNA at the 3′ end, and no more than three contiguousLNAs. The oligonucleotide may comprise a sequence with at least fiveLNAs, a LNA at the 5′ end, a LNA at the 3′ end, and no more than threecontiguous LNAs, wherein the sequence is at least 16 nucleotides inlength. The sequence can be substantially or completely complementary toa RNA, such as mRNA, or miRNA, wherein a substantially complementarysequence may have from 1 to 4 mismatches (e.g., 1 or 2 mismatches) withrespect to its target sequence. In one embodiment, the target sequenceis a miRNA, such that the oligonucleotide is a miRNA inhibitor, orantimiR. In one embodiment, the target sequence is a miR-92 sequence asprovided herein.

In yet another embodiment, the oligonucleotide of the present inventioncan comprise a sequence complementary to the seed region of a miRNA(e.g., miR-92), wherein the sequence comprises at least five LNAs. The“seed region of a miRNA” is the portion spanning bases 2 to 9 at the 5′end of the miRNA. The oligonucleotide comprising a sequencecomplementary to the seed region of a miRNA (e.g., miR-92), wherein thesequence comprises at least five LNAs, may comprise a LNA at the 5′ endor a LNA at the 3′ end, or both a LNA at the 5′ end and 3′ end. In oneembodiment, the oligonucleotide comprising at least 5 LNAs, a LNA at the5′ end and/or a LNA at the 3′ end, also has three or fewer consecutiveLNAs. In some embodiments, the sequence is at least 16 nucleotides inlength. The sequence complementary to the seed region of a miRNA can besubstantially complementary or completely complementary.

The oligonucleotides of the present invention may comprise one or morelocked nucleic acid (LNAs) residues, or “locked nucleotides.” Theoligonucleotide of the present invention can contain one or more lockednucleic acid (LNAs) residues, or “locked nucleotides.” Theoligonucleotides of the present invention may comprise one or morenucleotides containing other sugar or base modifications. The terms“locked nucleotide,” “locked nucleic acid unit,” “locked nucleic acidresidue,” “LNA” or “LNA unit” may be used interchangeably throughout thedisclosure and refer to a bicyclic nucleoside analogue. For instance,suitable oligonucleotide inhibitors can be comprised of one or more“conformationally constrained” or bicyclic sugar nucleosidemodifications (BSN) that confer enhanced thermal stability to complexesformed between the oligonucleotide containing BSN and theircomplementary target strand. LNAs are described, for example, in U.S.Pat. Nos. 6,268,490, 6,316,198, 6,403,566, 6,770,748, 6,998,484,6,670,461, and 7,034,133, all of which are hereby incorporated byreference in their entireties. LNAs are modified nucleotides orribonucleotides that contain an extra bridge between the 2′ and 4′carbons of the ribose sugar moiety resulting in a “locked” conformation,and/or bicyclic structure. In one embodiment, the oligonucleotidecontains one or more LNAs having the structure shown by structure Abelow. Alternatively or in addition, the oligonucleotide may contain oneor more LNAs having the structure shown by structure B below.Alternatively or in addition, the oligonucleotide contains one or moreLNAs having the structure shown by structure C below.

When referring to substituting a DNA or RNA nucleotide by itscorresponding locked nucleotide in the context of the present invention,the term “corresponding locked nucleotide” is intended to mean that theDNA/RNA nucleotide has been replaced by a locked nucleotide containingthe same naturally-occurring nitrogenous base as the DNA/RNA nucleotidethat it has replaced or the same nitrogenous base that is chemicallymodified. For example, the corresponding locked nucleotide of a DNAnucleotide containing the nitrogenous base C may contain the samenitrogenous base C or the same nitrogenous base C that is chemicallymodified, such as 5-methylcytosine.

The term “non-locked nucleotide” refers to a nucleotide different from alocked-nucleotide, i.e. the term “non-locked nucleotide” includes a DNAnucleotide, an RNA nucleotide as well as a modified nucleotide where abase and/or sugar is modified except that the modification is not alocked modification.

Other suitable locked nucleotides that can be incorporated in theoligonucleotides of the present invention include those described inU.S. Pat. Nos. 6,403,566 and 6,833,361, both of which are herebyincorporated by reference in their entireties.

In exemplary embodiments, the locked nucleotides have a 2′ to 4′methylene bridge, as shown in structure A, for example. In otherembodiments, the bridge comprises a methylene or ethylene group, whichmay be substituted, and which may or may not have an ether linkage atthe 2′ position.

Oligonucleotide inhibitors of the present invention may include modifiednucleotides that have a base modification or substitution. The naturalor unmodified bases in RNA are the purine bases adenine (A) and guanine(G), and the pyrimidine bases cytosine (C) and uracil (U) (DNA hasthymine (T)). Modified bases, also referred to as heterocyclic basemoieties, include other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines), 7-methylguanine and 7-methyladenine,2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.In certain embodiments, oligonucleotide inhibitors targeting miR-92comprise one or more BSN modifications (i.e., LNAs) in combination witha base modification (e.g. 5-methyl cytidine).

Oligonucleotide inhibitors of the present invention may includenucleotides with modified sugar moieties. Representative modified sugarsinclude carbocyclic or acyclic sugars, sugars having substituent groupsat one or more of their 2′, 3′ or 4′ positions and sugars havingsubstituents in place of one or more hydrogen atoms of the sugar. Incertain embodiments, the sugar is modified by having a substituent groupat the 2′ position. In additional embodiments, the sugar is modified byhaving a substituent group at the 3′ position. In other embodiments, thesugar is modified by having a substituent group at the 4′ position. Itis also contemplated that a sugar may have a modification at more thanone of those positions, or that an oligonucleotide inhibitor may haveone or more nucleotides with a sugar modification at one position andalso one or more nucleotides with a sugar modification at a differentposition.

The oligonucleotide may comprise, consist essentially of, or consist of,an antisense sequence to miR-92. In one embodiment, the oligonucleotidecomprises an antisense sequence directed to miR-92. For example, theoligonucleotide can comprise a sequence that is at least partiallycomplementary to a mature miR-92 sequence, e.g. at least about 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 6%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a humanmature miR-92 sequence. In one embodiment, the oligonucleotide inhibitoras provided herein comprises a sequence that is 100% or fullycomplementary to a mature miR-92 sequence. It is understood that thesequence of the oligonucleotide inhibitor is considered to becomplementary to miR-92 even if the oligonucleotide inhibitor sequenceincludes a modified nucleotide instead of a naturally-occurringnucleotide. For example, if a mature sequence of miR-92 comprises aguanosine nucleotide at a specific position, the oligonucleotideinhibitor may comprise a modified cytidine nucleotide, such as a lockedcytidine nucleotide or 2′-fluoro-cytidine, at the corresponding position

The term “about” as used herein is meant to encompass variations of+/−10% and more preferably +/−5%, as such variations are appropriate forpracticing the present invention.

In certain embodiments, the oligonucleotide comprises a nucleotidesequence that is completely complementary to a nucleotide sequence ofmiR-92. In particular embodiments, the oligonucleotide comprises,consists essentially of, or consists of the nucleotide sequencecomplementary to miR-92. In this context, “consists essentially of”includes the optional addition of nucleotides (e.g., one or two) oneither or both of the 5′ and 3′ ends, so long as the additionalnucleotide(s) do not substantially affect (as defined by an increase inIC50 of no more than 20%) the oligonucleotide's inhibition of the targetmiRNA activity in the dual luciferase assay or animal (e.g., mouse)model.

The oligonucleotide can generally have a nucleotide sequence designed totarget mature miR-92. The oligonucleotide may, in these or otherembodiments, also or alternatively be designed to target the pre- orpri-miRNA forms of miR-92. In certain embodiments, the oligonucleotidemay be designed to have a sequence containing from 1 to 5 (e.g., 1, 2,3, or 4) mismatches relative to the fully complementary (mature) miR-92sequence. In certain embodiments, such antisense sequences may beincorporated into shRNAs or other RNA structures containing stem andloop portions, for example.

The oligonucleotide can be from 8 to 20 nucleotides in length, from 15to 50 nucleotides in length, from 18 to 50 nucleotides in length, from10 to 18 nucleotides in length, or from 11 to 16 nucleotides in length.The oligonucleotide in some embodiments is about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17, orabout 18 nucleotides in length. In one embodiment, the present inventionprovides an oligonucleotide inhibitor of miR-92 that has a length of 11to 16 nucleotides. In various embodiments, the oligonucleotide inhibitortargeting miR-92 is 11, 12, 13, 14, 15, or 16 nucleotides in length. Inone embodiment, the oligonucleotide inhibitor of miR-92 has a length of12 nucleotides. In some embodiments, the oligonucleotide inhibitor ofmiR-92 is at least 16 nucleotides in length.

Generally, the number and position of LNA can be such that theoligonucleotide reduces miR-92 activity or function. In one embodiment,the number and position of LNAs is such that the oligonucleotide has anincreased efficacy relative to a control. In some embodiments, efficacyis a capacity for producing a beneficial or desired result (e.g.,clinical result). The beneficial or desired result can be a reduction,amelioration, or removal of a symptom or symptoms of a disease orcondition. The beneficial or desired result can be a inhibition,reduction, amelioration, or removal of the activity or function ofmiR-92. The increased efficacy can be increased in vivo, in vitro, or exvivo. The control can be an oligonucleotide containing the same sequenceas the oligonucleotide comprising LNAs as provided herein but nochemical modifications. The control can be an oligonucleotide containingthe same sequence as the oligonucleotide comprising LNAs as providedherein but a different chemical modification motif or pattern. Thecontrol can be an oligonucleotide containing the same sequence as theoligonucleotide comprising LNAs as provided herein but a differentnumber and/or position of LNAs. The control can be an oligonucleotidecontaining the same sequence as well as number and/or position of LNAs,but a different additional modification such as the presence of one ormore 5-methylcytosines.

The oligonucleotide as provided herein generally contains at least about2, at least about 3, at least about 4, at least about 5, at least about7, or at least about 9 LNAs, but in various embodiments is not fullycomprised of LNAs. Generally, the number and position of LNAs is suchthat the oligonucleotide reduces mRNA or miRNA function or activity. Incertain embodiments, the oligonucleotide does not contain a stretch ofnucleotides with more than four, or more than three, contiguous LNAs.For example, the oligonucleotide comprises no more than three contiguousLNAs. In these or other embodiments, the oligonucleotide can comprise aregion or sequence that is substantially or completely complementary toa miRNA seed region, in which the region or sequence comprises at leasttwo, at least three, at least four, or at least five locked nucleotides.

In certain embodiments, the oligonucleotide inhibitor contains at least1, at least 2, at least 3, at least 4, or at least 5 DNA nucleotides. Inone embodiment, the oligonucleotide inhibitor comprises at least oneLNA, wherein each non-locked nucleotide in the oligonucleotide inhibitoris a DNA nucleotide. In one embodiment, the oligonucleotide inhibitorcomprises at least two LNAs, wherein each non-locked nucleotide in theoligonucleotide inhibitor is a DNA nucleotide. In one embodiment, atleast the second nucleotide from the 5′ end of the oligonucleotideinhibitor is a DNA nucleotide. In one embodiment, at least 1, at least2, at least 3, at least 4, or at least 5 DNA nucleotides in anoligonucleotide as provided herein contains a nitrogenous base that ischemically modified. In one embodiment, the second nucleotide from the5′ end of an oligonucleotide inhibitor as provided herein contains anitrogenous base that is chemically modified. The chemically modifiednitrogenous base can be 5-methylcytosine. In one embodiment, the secondnucleotide from the 5′ end is a 5-methylcytosine. In one embodiment, anoligonucleotide inhibitor as provided herein comprises a5-methylcytosine at each LNA that is a cytosine.

In one embodiment, an oligonucleotide inhibitor of miR-92 as providedherein comprises a sequence of 12 to 16 nucleotides, wherein thesequence is at least partially or fully complementary to a maturesequence of miR-92, in which from the 5′ end to the 3′ end of theoligonucleotide, at least the first and last nucleotide positions areLNAs. In certain embodiments, the oligonucleotide inhibitor of miR-92has a length of 12 nucleotides. In certain embodiments, theoligonucleotide inhibitor of miR-92 has a length of 13 nucleotides. Incertain embodiments, the oligonucleotide inhibitor of miR-92 has alength of 14 nucleotides. In certain embodiments, the oligonucleotideinhibitor of miR-92 has a length of 15 nucleotides. In certainembodiments, the oligonucleotide inhibitor of miR-92 has a length of 16nucleotides. The oligonucleotide can have a full or partial (i.e., oneor more) phosphorothioate backbone. The oligonucleotide can furthercomprise any additional modification as provided herein including butnot limited to one or more chemically modified nitrogenous bases, a 5′and/or 3′ cap structure, a pendent lipophilic group and/or 2′ deoxy, 2′O-alkyl or 2′ halo modification(s). In certain embodiments, theoligonucleotide inhibitor of miR-92 comprising a sequence of from 12 to16 nucleotides comprises at least one nucleotide with a chemicallymodified nitrogenous base. The chemically modified nitrogenous base canbe a methylated base. In certain embodiments, the chemically modifiednitrogenous base is 5-methylcytosine. In one embodiment, each LNA thatis a cytosine is a 5-methylcytosine. In certain embodiments, anoligonucleotide inhibitor as provided herein comprising at least onenucleotide with a chemically modified nitrogenous base (e.g.,5-methylcytosine) shows increased efficacy as compared to the sameoligonucleotide inhibitor lacking the chemically modified nitrogenousbase. The increased efficacy can be an increased reduction or inhibitionof miR-92 function and/or activity. The increased efficacy can be invivo, ex vivo and/or in vitro.

In one embodiment, the oligonucleotide can comprise a sequence of 13 to16 nucleotides, in which from the 5′ end to the 3′ end of theoligonucleotide, positions 1, 6, 10, 11 and 13 are LNAs, and theremaining positions are non-locked nucleotides, wherein theoligonucleotide is at least partially complementary to a miRNA or a seedregion of a miRNA, in which the miRNA may in some embodiments, bemiR-92. The oligonucleotide can be fully complementary to the miRNA, inwhich the miRNA may in some embodiments, be miR-92. In some embodiments,at least one non-locked nucleotide comprises a nitrogenous base that ischemically modified. In certain embodiments, the oligonucleotideinhibitor comprises a nucleotide containing a chemically modifiednitrogenous base at a second nucleotide position from the 5′ end to the3′ end of the oligonucleotide. In certain embodiments, the secondnucleotide position is a cytosine and the chemically modifiednitrogenous base is a 5-methylcytosine. In one embodiment, the presenceof the chemically modified nitrogenous base(s) (e.g., 5-methylcytosine)in the oligonucleotide inhibitor has increased in vivo or in vitroefficacy as compared to an oligonucleotide with the same number and/orposition of LNAs but no chemically modified nitrogenous base (e.g.,5-methylcytosine). The increased efficacy can be an increased reductionof miRNA (e.g., miR-92) function and/or activity.

In another embodiment, the oligonucleotide can comprise at least 16nucleotides, in which from the 5′ end to the 3′ end of theoligonucleotide, positions 1, 3, 6, 8, 10, 11, 13, 14, and 16 are LNAs,and the remaining positions are non-locked nucleotides, theoligonucleotide is at least partially complementary to a miRNA or a seedregion of a miRNA, in which the miRNA may in some embodiments, bemiR-92. The oligonucleotide can be fully complementary to the miRNA, inwhich the miRNA may in some embodiments, be miR-92. In some embodiments,the second nucleotide from the 5′ end comprises a nitrogenous base thatis chemically modified (e.g. 5-methylcytosine). In one embodiment, thepresence of the chemically modified nitrogenous base(s) (e.g.,5-methylcytosine) in the oligonucleotide inhibitor has increased in vivoor in vitro efficacy as compared to an oligonucleotide with the samenumber and/or position of LNAs but no chemically modified nitrogenousbase (e.g., 5-methylcytosine). The increased efficacy can be anincreased reduction of miRNA (e.g., miR-92) function and/or activity.

In another embodiment, the oligonucleotide can comprise at least 16nucleotides, in which from the 5′ end to the 3′ end of theoligonucleotide, positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 are LNAs,and the remaining positions are non-locked nucleotides, theoligonucleotide is at least partially complementary to a miRNA or a seedregion of a miRNA, in which the miRNA may in some embodiments, bemiR-92. The oligonucleotide can be fully complementary to the miRNA, inwhich the miRNA may in some embodiments, be miR-92. In some embodiments,the second nucleotide from the 5′ end comprises a nitrogenous base thatis chemically modified (e.g. 5-methylcytosine). In one embodiment, thepresence of the chemically modified nitrogenous base(s) (e.g.,5-methylcytosine) in the oligonucleotide inhibitor has increased in vivoor in vitro efficacy as compared to an oligonucleotide with the samenumber and/or position of LNAs but no chemically modified nitrogenousbase (e.g., 5-methylcytosine). The increased efficacy can be anincreased reduction of miRNA (e.g., miR-92) function and/or activity.

In another embodiment, the oligonucleotide can comprise at least 16nucleotides, in which from the 5′ end to the 3′ end of theoligonucleotide, positions 1, 3, 6, 9, 10, 11, 13, 14, and 16 are LNAs,and the remaining positions are non-locked nucleotides, theoligonucleotide is at least partially complementary to a miRNA or a seedregion of a miRNA, in which the miRNA may in some embodiments, bemiR-92. The oligonucleotide can be fully complementary to the miRNA, inwhich the miRNA may in some embodiments, be miR-92. In some embodiments,the second nucleotide from the 5′ end comprises a nitrogenous base thatis chemically modified (e.g. 5-methylcytosine). In one embodiment, thepresence of the chemically modified nitrogenous base(s) (e.g.,5-methylcytosine) in the oligonucleotide inhibitor has increased in vivoor in vitro efficacy as compared to an oligonucleotide with the samenumber and/or position of LNAs but no chemically modified nitrogenousbase (e.g., 5-methylcytosine). The increased efficacy can be anincreased reduction of miRNA (e.g., miR-92) function and/or activity.

In some embodiments, the oligonucleotide is selected from Tables 1 or 2.In certain embodiments, the oligonucleotide is an oligonucleotideinhibitor selected from Table 2.

In some embodiments, an oligonucleotide inhibitor as provided herein(e.g., miR-92 oligonucleotide inhibitor) shows at least about 0.5%, atleast about 1%, at least about 2%, at least about 3%, at least about 4%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or at least about 99% greater inhibition of the function and/or activityof a target miRNA (e.g., miR-92) as compared to other inhibitors of thetarget miRNA (e.g., miR-92). The improvement or increase can be invitro, ex vivo and/or in vivo.

In some embodiments, an oligonucleotide inhibitor as provided herein(e.g., miR-92 oligonucleotide inhibitor) comprising a 5-methylcytosineproduces at least about 0.5%, at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 99% of anincrease or improvement in the reduction of function and/or activity ofa target miRNA (e.g., miR-92) as compared to an oligonucleotide with thesame nucleotide sequence as well as LNA/DNA pattern but lacking a5-methylcyotsine. The improvement or increase can be in vitro, ex vivoand/or in vivo. In some cases, all LNA cytosines in an oligonucleotideinhibitor as provided herein is a 5-methylcytosine LNA.

In some embodiments for non-locked nucleotides, the nucleotide maycontain a 2′ modification with respect to a 2′ hydroxyl. For example,the 2′ modification may be 2′ deoxy. Incorporation of 2′-modifiednucleotides in antisense oligonucleotides may increase resistance of theoligonucleotides to nucleases. Incorporation of 2′-modified nucleotidesin antisense oligonucleotides may increase their thermal stability withcomplementary RNA. Incorporation of 2′-modified nucleotides in antisenseoligonucleotides may increase both resistance of the oligonucleotides tonucleases and their thermal stability with complementary RNA. Variousmodifications at the 2′ positions may be independently selected fromthose that provide increased nuclease sensitivity, without compromisingmolecular interactions with the RNA target or cellular machinery. Suchmodifications may be selected on the basis of their increased potency invitro, ex vivo or in vivo. Exemplary methods for determining increasedpotency (e.g., IC50) for miR-92 inhibition are described herein,including, but not limited to, the dual luciferase assay and in vivomiR-92 abundance or target de-repression.

In some embodiments the 2′ modification may be independently selectedfrom O-alkyl (which may be substituted), halo, and deoxy (H).Substantially all, or all, nucleotide 2′ positions of the non-lockednucleotides may be modified in certain embodiments, e.g., asindependently selected from O-alkyl (e.g., O-methyl), halo (e.g.,fluoro), deoxy (H), and amino. For example, the 2′ modifications mayeach be independently selected from O-methyl (OMe) and fluoro (F). Inexemplary embodiments, purine nucleotides each have a 2′ OMe andpyrimidine nucleotides each have a 2′-F. In certain embodiments, fromone to about five 2′ positions, or from about one to about three 2′positions are left unmodified (e.g., as 2′ hydroxyls).

2′ modifications in accordance with the invention can also include smallhydrocarbon substituents. The hydrocarbon substituents include alkyl,alkenyl, alkynyl, and alkoxyalkyl, where the alkyl (including the alkylportion of alkoxy), alkenyl and alkynyl may be substituted orunsubstituted. The alkyl, alkenyl, and alkynyl may be C1 to C10 alkyl,alkenyl or alkynyl, such as Cl, C2, or C3. The hydrocarbon substituentsmay include one or two or three non-carbon atoms, which may beindependently selected from nitrogen (N), oxygen (O), and/or sulfur (S).The 2′ modifications may further include the alkyl, alkenyl, and alkynylas O-alkyl, O-alkenyl, and O-alkynyl.

Exemplary 2′ modifications in accordance with the invention can include2′-O-alkyl (C1-3 alkyl, such as 2′ OMe or 2′OEt), 2′-O-methoxyethyl(2′-0-MOE), 2′-0-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O—NMA) substitutions.

In certain embodiments, the oligonucleotide contains at least one2′-halo modification (e.g., in place of a 2′ hydroxyl), such as2′-fluoro, 2′-chloro, 2′-bromo, and 2′-iodo. In some embodiments, the 2′halo modification is fluoro. The oligonucleotide may contain from 1 toabout 5 2′-halo modifications (e.g., fluoro), or from 1 to about 32′-halo modifications (e.g., fluoro). In some embodiments, theoligonucleotide contains all 2′-fluoro nucleotides at non-lockedpositions, or 2′-fluoro on all non-locked pyrimidine nucleotides. Incertain embodiments, the 2′-fluoro groups are independently di-, tri-,or un-methylated.

The oligonucleotide may have one or more 2′-deoxy modifications (e.g., Hfor 2′ hydroxyl), and in some embodiments, contains from 2 to about 102′-deoxy modifications at non-locked positions, or contains 2′ deoxy atall non-locked positions.

In exemplary embodiments, the oligonucleotide contains 2′ positionsmodified as 2′OMe in non-locked positions. Alternatively, non-lockedpurine nucleotides can be modified at the 2′ position as 2′OMe, withnon-locked pyrimidine nucleotides modified at the 2′ position as2′-fluoro.

In certain embodiments, the oligonucleotide further comprises at leastone terminal modification or “cap.” The cap may be a 5′ and/or a 3′-capstructure. The terms “cap” or “end-cap” include chemical modificationsat either terminus of the oligonucleotide (with respect to terminalribonucleotides), and includes modifications at the linkage between thelast two nucleotides on the 5′ end and the last two nucleotides on the3′ end. The cap structure as described herein may increase resistance ofthe oligonucleotide to exonucleases without compromising molecularinteractions with the miRNA target (i.e. miR-92) or cellular machinery.Such modifications may be selected on the basis of their increasedpotency in vitro or in vivo. The cap can be present at the 5′-terminus(5′-cap) or at the 3′-terminus (3′-cap) or can be present on both ends.In certain embodiments, the 5′- and/or 3′-cap is independently selectedfrom phosphorothioate monophosphate, abasic residue (moiety),phosphorothioate linkage, 4′-thio nucleotide, carbocyclic nucleotide,phosphorodithioate linkage, inverted nucleotide or inverted abasicmoiety (2′-3′ or 3′-3′), phosphorodithioate monophosphate, andmethylphosphonate moiety. The phosphorothioate or phosphorodithioatelinkage(s), when part of a cap structure, are generally positionedbetween the two terminal nucleotides on the 5′ end and the two terminalnucleotides on the 3′ end.

In certain embodiments, the oligonucleotide has at least one terminalphosphorothioate monophosphate. The phosphorothioate monophosphate maysupport a higher potency by inhibiting the action of exonucleases. Thephosphorothioate monophosphate may be at the 5′ and/or 3′ end of theoligonucleotide. A phosphorothioate monophosphate is defined by thefollowing structures, where B is base, and R is a 2′ modification asdescribed above:

Where the cap structure can support the chemistry of a lockednucleotide, the cap structure may incorporate a LNA as described herein.

Phosphorothioate linkages may be present in some embodiments, such asbetween the last two nucleotides on the 5′ and the 3′ end (e.g., as partof a cap structure), or as alternating with phosphodiester bonds. Inthese or other embodiments, the oligonucleotide may contain at least oneterminal abasic residue at either or both the 5′ and 3′ ends. An abasicmoiety does not contain a commonly recognized purine or pyrimidinenucleotide base, such as adenosine, guanine, cytosine, uracil orthymine. Thus, such abasic moieties lack a nucleotide base or have othernon-nucleotide base chemical groups at the 1′ position. For example, theabasic nucleotide may be a reverse abasic nucleotide, e.g., where areverse abasic phosphoramidite is coupled via a 5′ amidite (instead of3′ amidite) resulting in a 5′-5′ phosphate bond. The structure of areverse abasic nucleoside for the 5′ and the 3′ end of a polynucleotideis shown below.

The oligonucleotide may contain one or more phosphorothioate linkages.Phosphorothioate linkages can be used to render oligonucleotides moreresistant to nuclease cleavage. For example, the polynucleotide may bepartially phosphorothioate-linked, for example, phosphorothioatelinkages may alternate with phosphodiester linkages. In certainembodiments, however, the oligonucleotide is fullyphosphorothioate-linked. In other embodiments, the oligonucleotide hasfrom one to five or one to three phosphate linkages.

In some embodiments, the nucleotide has one or more carboxamido-modifiedbases as described in PCT/US11/59588, which is hereby incorporated byreference, including with respect to all exemplary pyrimidinecarboxamido modifications disclosed therein with heterocyclicsubstituents.

The synthesis of oligonucleotides, including modified polynucleotides,by solid phase synthesis is well known and is reviewed in Caruthers etal., Nucleic Acids Symp. Ser. 7:215-23 (1980).

In some embodiments, the oligonucleotide comprises a sequence selectedfrom Tables 1 and 2, in which “+” or “1” indicates the nucleotide is aLNA; “d” indicates the nucleotide is a DNA; “s” indicates aphosphorothioate linkage between the two nucleotides; and “mdC”indicates the nucleotide is a 5-methyl cytosine DNA:

TABLE 1 MiR-92 Inhibitors SEQ ID Sequence (5′ to 3′) NO. Alias(second line of sequence is with linkages notation) SEQ 92a_LNA_16_PS+CC+GGG+AC+AA+G+TG+C+AA+T IDlCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 7 SEQ92a_LNA_16_1 +CCGG+G+AC+AA+G+TG+CA+A+T IDlCs;dCs;dGs;dGs;lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 8 SEQ92a_LNA_16_4 +CC+GGG+ACA+A+G+TG+C+AA+T IDlCs;dCs;lGs;dGs;dGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 9

TABLE 2 Additional MiR-92 Inhibitors SEQ ID Sequence (5′ to 3′) NO.Alias (second line of sequence is with linkages notation) SEQ ID92a_Tiny_LNA lAs;lGs;lTs;lGs;lCs;lAs;lAs;lT; NO: 10 +A+G+T+G+C+A+A+TSEQ ID 92a_LNA_16_2lCs;dCs;lGs;lGs;dGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 11+CC+G+GGA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_16_3lCs;dCs;dGs;lGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 12+CCG+G+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_16_5lCs;dCs;lGs;dGs;lGs;dAs;lCs;dAs;lAs;dGs;lTs;dGs;lCs;dAs;lAs;lT NO: 13+CC+GG+GA+CA+AG+TG+CA+A+T SEQ ID 92a_LNA_16_6lCs;lCs;dGs;lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 14+C+CG+GG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_16_7lCs;dCs;dGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;lTs;lGs;lCs;lAs;lAs;lT NO: 15+CCGG+GAC+AAG+T+G+C+A+A+T SEQ ID 92a_LNA_16_8lCs;dCs;dGs;lGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 16+CCG+GG+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_16_9lCs;dCs;lGs;dGs;lGs;dAs;dCs;lAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 17+CC+GG+GAC+AA+GT+G+C+AA+T SEQ ID 92a_LNA16_10lCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 18+CC+GGG+AC+AA+G+TGC+A+A+T SEQ ID 92a_LNA_16_11lCs;lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 19+C+C+GGG+AC+AAG+TGC+A+A+T SEQ ID 92a_LNA_16_12lCs;lCs;lGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 20+C+C+GG+GAC+AAG+TGC+A+A+T SEQ ID 92a_LNA_16_13lCs;lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 21+C+C+GGG+AC+AAG+TG+C+AA+T SEQ ID 92a_LNA_16_14lCs;dCs;lGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 22+CC+GG+GA+CAA+G+TG+CA+A+T SEQ ID 92a_LNA_16_15lCs;lCs;dGs;dGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;lCs;dAs;dAs;lT NO: 23+C+CGG+GA+CA+A+GT+G+CAA+T SEQ ID 92a_LNA_16_16lCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;lAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 24+CC+GGG+AC+A+AG+TG+C+AA+T SEQ ID 92a_LNA_16_17lCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 25+CC+GGG+AC+AA+GT+G+C+AA+T SEQ ID 92a_LNA_16_18lCs;dCs;lGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 26+CC+GG+GA+CAA+G+TGC+A+A+T SEQ ID 92a_LNA_16_19lCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;lAs;lT NO: 27+CC+GGG+AC+AAG+TG+C+A+A+T SEQ ID 92a_LNA_16_20lCs;dCs;lGs;dGs;lGs;dAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 28+CC+GG+GAC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_16_21lCs;lCs;dGs;dGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 29+C+CGG+GA+C+AA+GTG+CA+A+T SEQ ID 92a_LNA_16_22lCs;dCs;lGs;lGs;dGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 30+CC+G+GGA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_16_23lCs;dCs;dGs;dGs;lGs;lAs;lCs;dAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 31+CCGG+G+A+CAA+G+TG+CA+A+T SEQ ID 92a_LNA_16_24lCs;dCs;dGs;lGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 32+CCG+G+GA+CAA+GT+G+C+AA+T SEQ ID 92a_LNA_16_25lCs;dCs;dGs;lGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 33+CCG+G+GA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_15_1lCs;dGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 34+CGGG+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_15_2lCs;dGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 35+CGGG+AC+AA+G+TG+CA+A+T SEQ ID 92a_LNA_15_3lCs;dGs;lGs;dGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 36+CG+GGA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_15_4lCs;dGs;dGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 37+CGG+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_15_5lCs;dGs;dGs;dGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 38+CGGG+ACA+A+G+TG+C+AA+T SEQ ID 92a_LNA_15_6lCs;lGs;dGs;lGs;dAs;lCs;dAs;lAs;dGs;lTs;dGs;lCs;dAs;dAs;lT NO: 39+C+GG+GA+CA+AG+TG+CAA+T SEQ ID 92a_LNA_15_7lCs;dGs;lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 40+CG+GG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_15_8lCs;dGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;dTs;lGs;lCs;lAs;lAs;lT NO: 41+CGG+GAC+AAGT+G+C+A+A+T SEQ ID 92a_LNA_15_9lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;dCs;lAs;lAs;lT NO: 42+C+GGG+AC+AA+GTGC+A+A+T SEQ ID 92a_LNA_15_10lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 43+C+GGG+AC+AAG+TGC+A+A+T SEQ ID 92a_LNA_15_11lCs;lGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 44+C+GG+GAC+AAG+TGC+A+A+T SEQ ID 92a_LNA_15_12lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 45+C+GGG+AC+AAG+TG+C+AA+T SEQ ID 92a_LNA_15_13lCs;lGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 46+C+GG+GA+CAA+GTG+CA+A+T SEQ ID 92a_LNA_15_14lCs;dGs;dGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;lCs;dAs;dAs;lT NO: 47+CGG+GA+CA+A+GT+G+CAA+T SEQ ID 92a_LNA_15_15lCs;lGs;dGs;dGs;lAs;dCs;dAs;lAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 48+C+GGG+ACA+AG+TG+C+AA+T SEQ ID 92a_LNA_15_16lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 49+C+GGG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_15_17lCs;lGs;dGs;dGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 50+C+GGGA+CAA+G+TGC+A+A+T SEQ ID 92a_LNA_15_18lCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;dAs;lAs;lT NO: 51+C+GGG+AC+AAG+TG+CA+A+T SEQ ID 92a_LNA_15_19lCs;lGs;dGs;lGs;dAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;lAs;dAs;lT NO: 52+C+GG+GAC+AA+GTG+C+AA+T SEQ ID 92a_LNA_15_20lCs;dGs;dGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 53+CGG+GA+C+AA+GTG+CA+A+T SEQ ID 92a_LNA_15_21lCs;dGs;lGs;dGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 54+CG+GGA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_15_22lCs;dGs;dGs;lGs;lAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 55+CGG+G+A+CAA+GTG+CA+A+T SEQ ID 92a_LNA_15_23lCs;dGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 56+CGG+GA+CAA+GT+G+C+AA+T SEQ ID 92a_LNA_15_24lCs;dGs;dGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 57+CGG+GA+C+AA+GT+GC+AA+T SEQ IDlCs;dCs;lGs;lGs;lGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;dAs;lT NO: 58+CC+G+G+G+AC+AA+GTG+CAA+T SEQ IDlCs;dCs;lGs;lGs;dGs;lAs;dCs;lAs;lAs;lGs;dTs;dGs;lCs;dAs;lAs;dT NO: 59+CC+G+GG+AC+A+A+GTG+CA+AT SEQ IDlCs;dCs;lGs;lGs;lGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;dT NO: 60+CC+G+G+G+AC+AA+GTG+CA+AT SEQ IDlCs;dCs;dGs;dGs;lGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 61+CCGG+G+ACA+A+G+TG+CA+A+T SEQ IDlCs;dCs;dGs;dGs;lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 62+CCGG+G+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdCs;dGs;dGs;lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 63+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs;mdCs;lGs;dGs;dGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 64+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs;mdCOGs;dGs;dGs;lAs;dCs;lAs;dAs;lGOTs;dGs;lCs;lAs;dAs;lT NO: 65+CC+GGG+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_14_1lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 66+GGG+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_14_2lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 67+GGG+AC+AA+G+TG+CA+A+T SEQ ID 92a_LNA_14_3lGs;lGs;dGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 68+G+GGA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_14_4lGs;dGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 69+GG+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_14_5lGs;dGs;dGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 70+GGG+ACA+A+G+TG+C+AA+T SEQ ID 92a_LNA_14_6lGs;dGs;lGs;dAs;lCs;dAs;lAs;dGs;lTs;dGs;lCs;dAs;dAs;lT NO: 71+GG+GA+CA+AG+TG+CAA+T SEQ ID 92a_LNA_14_7lGs;lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 72+G+GG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_14_8lGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;dTs;lGs;lCs;lAs;lAs;lT NO: 73+GG+GAC+AAGT+G+C+A+A+T SEQ ID 92a_LNA_14_9lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;dCs;lAs;lAs;lT NO: 74+GGG+AC+AA+GTGC+A+A+T SEQ ID 92a_LNA_14_10lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 75+GGG+AC+AAG+TGC+A+A+T SEQ ID 92a_LNA_14_11lGs;dGs;lGs;dAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 76+GG+GAC+AAG+TGC+A+A+T SEQ ID 92a_LNA_14_12lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 77+GGG+AC+AAG+TG+C+AA+T SEQ ID 92a_LNA_14_13lGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 78+GG+GA+CAA+GTG+CA+A+T SEQ ID 92a_LNA_14_14lGs;dGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;lCs;dAs;dAs;lT NO: 79+GG+GA+CA+A+GT+G+CAA+T SEQ ID 92a_LNA_14_15lGs;dGs;dGs;lAs;dCs;dAs;lAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 80+GGG+ACA+AG+TG+C+AA+T SEQ ID 92a_LNA_14_16lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 81+GGG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_14_17lGs;dGs;dGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 82+GGGA+CAA+G+TGC+A+A+T SEQ ID 92a_LNA_14_18lGs;dGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;dAs;lAs;lT NO: 83+GGG+AC+AAG+TG+CA+A+T SEQ ID 92a_LNA_14_19lGs;dGs;lGs;dAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;lAs;dAs;lT NO: 84+GG+GAC+AA+GTG+C+AA+T SEQ ID 92a_LNA_14_20lGs;dGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 85+GG+GA+C+AA+GTG+CA+A+T SEQ ID 92a_LNA_14_21lGs;lGs;dGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 86+G+GGA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_14_22lGs;dGs;lGs;lAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 87+GG+G+A+CAA+GTG+CA+A+T SEQ ID 92a_LNA_14_23lGs;dGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 88+GG+GA+CAA+GT+G+C+AA+T SEQ ID 92a_LNA_14_24lGs;dGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 89+GG+GA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_13_1lGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 90+GG+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_13_2lGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 91+GG+AC+AA+G+TG+CA+A+T SEQ ID 92a_LNA_13_3lGs;dGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 92+GGA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_13_4lGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 93+G+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_13_5lGs;dGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 94+GG+ACA+A+G+TG+C+AA+T SEQ ID 92a_LNA_13_6lGs;lGs;dAs;lCs;dAs;lAs;dGs;lTs;dGs;lCs;dAs;dAs;lT NO: 95+G+GA+CA+AG+TG+CAA+T SEQ ID 92a_LNA_13_7lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 96+GG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_13_8lGs;lGs;dAs;dCs;lAs;dAs;dGs;dTs;lGs;lCs;lAs;lAs;lT NO: 97+G+GAC+AAGT+G+C+A+A+T SEQ ID 92a_LNA_13_9lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;dCs;lAs;lAs;lT NO: 98+GG+AC+AA+GTGC+A+A+T SEQ ID 92a_LNA_13_10lGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 99+GG+AC+AAG+TGC+A+A+T SEQ ID 92a_LNA_13_11lGs;lGs;dAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 100+G+GAC+AAG+TGC+A+A+T SEQ ID 92a_LNA_13_12lGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 101+GG+AC+AAG+TG+C+AA+T SEQ ID 92a_LNA_13_13lGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 102+G+GA+CAA+GTG+CA+A+T SEQ ID 92a_LNA_13_14lGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;lCs;dAs;dAs;lT NO: 103+G+GA+CA+A+GT+G+CAA+T SEQ ID 92a_LNA_13_15lGs;dGs;lAs;dCs;dAs;lAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 104+GG+ACA+AG+TG+C+AA+T SEQ ID 92a_LNA_13_16lGs;dGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 105+GG+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_13_17lGs;dGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 106+GGA+CAA+G+TGC+A+A+T SEQ ID 92a_LNA_13_18lGs;dGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;dAs;lAs;lT NO: 107+GG+AC+AAG+TG+CA+A+T SEQ ID 92a_LNA_13_19lGs;lGs;dAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;lAs;dAs;lT NO: 108+G+GAC+AA+GTG+C+AA+T SEQ ID 92a_LNA_13_20lGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 109+G+GA+C+AA+GTG+CA+A+T SEQ ID 92a_LNA_13_21lGs;dGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 110+GGA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_13_22lGs;lGs;lAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 111+G+G+A+CAA+GTG+CA+A+T SEQ ID 92a_LNA_13_23lGs;lGs;dAs;lCs;dAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 112+G+GA+CAA+GT+G+C+AA+T SEQ ID 92a_LNA_13_24lGs;lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 113+G+GA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_12_1lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 114+G+AC+AA+G+TG+C+AA+T SEQ ID 92a_LNA_12_2lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 115+G+AC+AA+G+TG+CA+A+T SEQ ID 92a_LNA_12_3lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 116+GA+C+AA+GT+GC+AA+T SEQ ID 92a_LNA_12_4lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 117+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_12_5lGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 118+G+ACA+A+G+TG+C+AA+T SEQ ID 92a_LNA_12_6lGs;dAs;lCs;dAs;lAs;dGs;lTs;dGs;lCs;dAs;dAs;lT NO: 119+GA+CA+AG+TG+CAA+T SEQ ID 92a_LNA_12_7lGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 120+G+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_12_8lGs;dAs;dCs;lAs;dAs;dGs;dTs;lGs;lCs;lAs;lAs;lT NO: 121+GAC+AAGT+G+C+A+A+T SEQ ID 92a_LNA_12_9lGs;lAs;dCs;lAs;dAs;lGs;dTs;dGs;dCs;lAs;lAs;lT NO: 122+G+AC+AA+GTGC+A+A+T SEQ ID 92a_LNA_12_10lGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 123+G+AC+AAG+TGC+A+A+T SEQ ID 92a_LNA_12_11lGs;dAs;dCs;lAs;dAs;dGs;lTs;dGs;dCs;lAs;lAs;lT NO: 124+GAC+AAG+TGC+A+A+T SEQ ID 92a_LNA_12_12lGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 125+G+AC+AAG+TG+C+AA+T SEQ ID 92a_LNA_12_13lGs;dAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 126+GA+CAA+GTG+CA+A+T SEQ ID 92a_LNA_12_14lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;lCs;dAs;dAs;lT NO: 127+GA+CA+A+GT+G+CAA+T SEQ ID 92a_LNA_12_15lGs;lAs;dCs;dAs;lAs;dGs;lTs;dGs;lCs;lAs;dAs;lT NO: 128+G+ACA+AG+TG+C+AA+T SEQ ID 92a_LNA_12_16lGs;lAs;dCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 129+G+AC+AA+GT+GC+AA+T SEQ ID 92a_LNA_12_17lGs;dAs;lCs;dAs;dAs;lGs;lTs;dGs;dCs;lAs;lAs;lT NO: 130+GA+CAA+G+TGC+A+A+T SEQ ID 92a_LNA_12_18lGs;lAs;dCs;lAs;dAs;dGs;lTs;dGs;lCs;dAs;lAs;lT NO: 131+G+AC+AAG+TG+CA+A+T SEQ ID 92a_LNA_12_19lGs;dAs;dCs;lAs;dAs;lGs;dTs;dGs;lCs;lAs;dAs;lT NO: 132+GAC+AA+GTG+C+AA+T SEQ ID 92a_LNA_12_20lGs;dAs;lCs;lAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 133+GA+C+AA+GTG+CA+A+T SEQ ID 92a_LNA_12_21lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 134+GA+CA+A+GT+GC+AA+T SEQ ID 92a_LNA_12_22lGs;lAs;lCs;dAs;dAs;lGs;dTs;dGs;lCs;dAs;lAs;lT NO: 135+G+A+CAA+GTG+CA+A+T SEQ ID 92a_LNA_12_23lGs;dAs;lCs;dAs;dAs;lGs;dTs;lGs;lCs;lAs;dAs;lT NO: 136+GA+CAA+GT+G+C+AA+T SEQ ID 92a_LNA_12_24lGs;dAs;lCs;lAs;dAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 137+GA+C+AA+GT+GC+AA+T SEQ IDlCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lC;lA;dAJT NO: 138+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dG;lC;lA;dAJT NO: 139+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;dCs;lGs;dGs;dGs;lAs;dCs;lAs;dA;lG;lT;dG;lC;lA;dAJT NO: 140+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;dC;lGs;dG;dGs;lA;dCs;lA;dAs;lG;lTs;dG;lCs;lA;dAs;lT NO: 141+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;dC;lG;dGs;dG;lA;dCs;lA;dA;lGs;lT;dG;lCs;lA;dAJT NO: 142+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlC;dC;lG;dG;dG;lA;dC;lA;dA;lG;lT;dG;lC;lA;dA;lT NO: 143+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lC;lA;dAJT NO: 144+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdCs;lGs;dGs;dGs;lAs;dCs;lAs;dAs;lGs;lTs;dG;lC;lA;dAJT NO: 145+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdCs;lGs;dGs;dGs;lAs;dCs;lAs;dA;lG;lT;dG;lC;lA;dAJT NO: 146+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdC;lGs;dG;dGs;lA;dCs;lA;dAs;lG;lTs;dG;lCs;lA;dAs;lT NO: 147+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs;mdC;lG;dGs;dGJA;dCs;lA;dA;lGs;lT;dG;lCs;lA;dAJT NO: 148+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlC;mdC;lG;dG;dG;lA;dC;lA;dA;lG;lT;dG;lC;lA;dA;lT NO: 149+CC+GGG+AC+AA+G+TG+C+AA+T SEQ IDlCs.dmCs.dGs.dGs.lGslAs.dCslAs.dAs.lGs.lTs.dGs.lC.dA.lA.lT NO: 150+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs.dmCs.dGs.dGs.lGslAs.dCslAs.dAs.lGs.lTs.dG.lC.dA.lA.lT NO: 151+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs.dmCs.dGs.dGs.lGs.lAs.dCslAs.dA.lGlT.dG.lC.dAJAJT NO: 152+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs.dmC.dGs.dG.lGslA.dCs.lA.dAs.lG.lTs.dG.lCs.dA.lAs.lT NO: 153+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs.dmC.dG.dGs.lGJA.dCs.lA.dAlGs.lT.dG.lCs.dAJAJT NO: 154+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlC.dmC.dG.dG.lG.lA.dC.lA.dA.lG.lT.dG.lC.dA.lA.lT NO: 155+CCGG+G+AC+AA+G+TG+CA+A+T SEQ IDlCs.dmCs.lGs.dGs.dGs.lAs.dCs.dAslAslGs.lTs.dGs.lC.lA.dAJT NO: 156+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs.dmCs.lGs.dGs.dGs.lAs.dCs.dAslAslGs.lTs.dG.lC.lA.dA.lT NO: 157+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs.dmCs.lGs.dGs.dGs.lAs.dCs.dAs.lA.lGlT.dG.lC.lA.dA.lT NO: 158+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs.dmC.lGs.dG.dGs.lA.dCs.dAlAs.lG.lTs.dG.lCs.lA.dAs.lT NO: 159+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs.dmC.lG.dGs.dG.lA.dCs.dA.lA.lGs.lT.dG.lCs.lA.dA.lT NO: 160+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlC.dmC.lG.dG.dG.lA.dC.dA.lA.lG.lT.dG.lC.lA.dA.lT NO: 161+CC+GGG+ACA+A+G+TG+C+AA+T SEQ IDlCs;dCs;dGs;dGs;lGs;lAs;dCs;dAs;lAs;lGs;lTs;dGs;lCs;dAs;lAs;lT NO: 162+CCGG+G+ACA+A+G+TG+CA+A+T SEQ IDlCs;dCs;dGs;dGs;lGs;lAs;dCs;lAs;dAs;lGs;lTs;dGs;lCs;lAs;dAs;lT NO: 163+CCGG+G+AC+AA+G+TG+C+AA+T SEQ IDlCs;dCs;dGs;lGs;lGs;dAs;lCs;dAs;lAs;lGs;dTs;lGs;dCs;lAs;dAs;lT NO: 164+CCG+G+GA+CA+A+GT+GC+AA+T

In one embodiment, the oligonucleotide comprises a sequence selectedfrom Tables 1 and 2, and comprises at least one non-locked nucleotidethat is 2′ O-alkyl or 2′ halo modified. In some embodiments, theoligonucleotide comprises at least one LNA that has a 2′ to 4′ methylenebridge. In some embodiments, the oligonucleotide has a 5′ cap structure,3′ cap structure, or 5′ and 3′ cap structure. In yet other embodiments,the oligonucleotide comprises a pendent lipophilic group.

The oligonucleotide may be incorporated within a variety ofmacromolecular assemblies or compositions. Such complexes for deliverymay include a variety of liposomes, nanoparticles, and micelles,formulated for delivery to a patient. The complexes may include one ormore fusogenic or lipophilic molecules to initiate cellular membranepenetration. Such molecules are described, for example, in U.S. Pat. No.7,404,969 and U.S. Pat. No. 7,202,227, which are hereby incorporated byreference in their entireties. Alternatively, the oligonucleotide mayfurther comprise a pendant lipophilic group to aid cellular delivery,such as those described in WO 2010/129672, which is hereby incorporatedby reference.

The present invention also provides a method for delivering anoligonucleotide disclosed herein to a cell (e.g., as part of acomposition or formulation described herein) for reducing or inhibitingactivity or function of miR-92 in the cell. In one embodiment, theoligonucleotide comprises sequence at least partially complementary tomiR-92. In one embodiment, the oligonucleotide is selected from Tables 1or 2. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a cardiac or muscle cell. In some embodiments,the cell is involved in wound healing. In some embodiments, the cell isa fibrocyte, fibroblast, keratinocyte or endothelial cell. In yet otherembodiments, the cell is in vivo or ex vivo.

Also provided herein is a method for treating, ameliorating, orpreventing the progression of a condition in a subject comprisingadministering a pharmaceutical composition comprising an oligonucleotidedisclosed herein. The method generally comprises administering theoligonucleotide or composition comprising the same to a subject. Theterm “subject” or “patient” refers to any vertebrate including, withoutlimitation, humans and other primates (e.g., chimpanzees and other apesand monkey species), farm animals (e.g., cattle, sheep, pigs, goats andhorses), domestic mammals (e.g., dogs and cats), laboratory animals(e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g.,domestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like). In some embodiments,the subject is a mammal. In other embodiments, the subject is a human.The subject may have a condition associated with, mediated by, orresulting from, expression of miR-92.

In one embodiment, a method of promoting angiogenesis in a subjectcomprises administering to the subject an oligonucleotide disclosedherein. In one embodiment, the oligonucleotide comprises sequence atleast partially complementary to miR-92. In one embodiment, theoligonucleotide is selected from Tables 1 or 2. In some embodiments, thesubject suffers from ischemia, myocardial infarction, chronic ischemicheart disease, peripheral or coronary artery occlusion, ischemicinfarction, stroke, atherosclerosis, acute coronary syndrome, coronaryartery disease, carotid artery disease, diabetes, chronic wound(s), orperipheral vascular disease (e.g., peripheral artery disease).

In one embodiment, a method of promoting wound healing in a subjectcomprises administering to the subject a miR-92 inhibitor, such as anoligonucleotide disclosed herein. In one embodiment, the oligonucleotidecomprises sequence at least partially complementary to miR-92. In oneembodiment, the oligonucleotide is selected from Tables 1 or 2. In someembodiments, the subject suffers from diabetes. In some embodiments,healing of a chronic wound, diabetic foot ulcer, venous stasis leg ulceror pressure sore is promoted by administration of a miR-92 inhibitor.

In one embodiment, administration of a miR-92 inhibitor as providedherein provides at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% improvement in wound re-epithelialization or wound closureas compared to a wound not administered the miR-92 inhibitor. In someembodiments, administration of a miR-92 inhibitor as provided hereinprovides at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% more granulation tissue formation or neovascularization as comparedto a wound not administered the miR-92 inhibitor.

In one embodiment, administration of a miR-92 inhibitor as providedherein provides at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% improvement in wound re-epithelialization or wound closureas compared to a wound administered an agent known in the art fortreating wounds. In some embodiments, administration of a miR-92inhibitor as provided herein provides at least about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% more granulation tissue formation orneovascularization as compared to a wound administered an agent known inthe art for treating wounds. The agent can be a growth factor such asfor example platelet derived growth factor (PDGF) and/or vascularendothelial growth factor (VEGF).

Also provided herein is an agonist of miR-92. An agonist of miR-92 canbe an oligonucleotide comprising a mature miR-92 sequence. In someembodiments, the oligonucleotide comprises the sequence of the pri-miRNAor pre-miRNA sequence for miR-92. The oligonucleotide comprising themature miR-92, pre-miR-92, or pri miR-92 sequence can be single strandedor double stranded. In one embodiment, the miR-92 agonist can be about15 to about 50 nucleotides in length, about 18 to about 30 nucleotidesin length, about 20 to about 25 nucleotides in length, or about 10 toabout 14 nucleotides in length. The miR-92 agonist can be at least about75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to themature, pri-miRNA or pre-miRNA sequence of miR-92. The miR-92 agonistthat is a oligonucleotide can contain one or more chemicalmodifications, such as locked nucleic acids, peptide nucleic acids,sugar modifications, such as 2′-O-alkyl (e.g. 2′-O-methyl,2′-O-methoxyethyl), 2′-fluoro, and 4′ thio modifications, and backbonemodifications, such as one or more phosphorothioate, morpholino, orphosphonocarboxylate linkages. In one embodiment, the oligonucleotidecomprising a miR-92 sequence is conjugated to cholesterol. Theoligonucleotide comprising a miR-92 sequence can be expressed in vivofrom a vector and/or operably linked to a promoter. In anotherembodiment, the agonist of miR-92 can be an agent distinct from miR-92that acts to increase, supplement, or replace the function of miR-92.

The present invention further provides pharmaceutical compositionscomprising an oligonucleotide disclosed herein. Where clinicalapplications are contemplated, pharmaceutical compositions can beprepared in a form appropriate for the intended application. Generally,this can entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

In one embodiment, the pharmaceutical composition comprises an effectivedose of a miR-92 inhibitor and a pharmaceutically acceptable carrier.For instance, the pharmaceutical composition comprises an effective doseor amount of an oligonucleotide of the present invention or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent. The oligonucleotide canbe selected from Tables 1 and 2.

In some embodiments, an “effective dose” is an amount sufficient toaffect a beneficial or desired clinical result. An “effective dose” canbe an amount sufficient or required to substantially reduce, eliminateor ameliorate a symptom or symptoms of a disease and/or condition. Thiscan be relative to an untreated subject. An “effective dose” can be anamount sufficient or required to slow, stabilize, prevent, or reduce theseverity of a pathology in a subject. This can be relative to anuntreated subject. An effective dose of an oligonucleotide disclosedherein may be from about 0.001 mg/kg to about 100 mg/kg, about 0.01mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1mg/kg to about 10 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5mg/kg to about 25 mg/kg. In some embodiments, an effective dose is anamount of oligonucleotide applied to a wound area. In some embodiments,an effective dose is about 0.01 mg/cm² wound area to about 50 mg/cm²wound area mg/cm² wound area, about 0.02 mg/cm² wound area to about 20mg/cm² wound area, about 0.1 mg/cm² wound area to about 10 mg/cm² woundarea, about 1 mg/cm² wound area to about 10 mg/cm² wound area, about 2.5mg/cm² wound area to about 50 mg/cm² wound area, or about 5 mg/cm² woundarea to about 25 mg/cm² wound area, or about 0.05 to about 25 mg/cm²wound area. The precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, and nature of the oligonucleotide (e.g.melting temperature, LNA content, etc.). Therefore, dosages can bereadily ascertained by those of ordinary skill in the art from thisdisclosure and the knowledge in the art. In some embodiments, themethods comprise administering an effective dose of the pharmaceuticalcomposition 1, 2, 3, 4, 5, or 6 times a day. In some embodiments,administration is 1, 2, 3, 4, 5, 6, or 7 times a week. In otherembodiments, administration is biweekly or monthly.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes, may beused as delivery vehicles for the oligonucleotide inhibitors of miR-92function. Commercially available fat emulsions that are suitable fordelivering the nucleic acids of the invention to cardiac and skeletalmuscle tissues include Intralipid™ Liposyn™, Liposyn™ II, Liposyn™ III,Nutrilipid, and other similar lipid emulsions. A preferred colloidalsystem for use as a delivery vehicle in vivo is a liposome (i.e., anartificial membrane vesicle). The preparation and use of such systems iswell known in the art. Exemplary formulations are also disclosed in U.S.Pat. Nos. 5,981,505; 6,217,900 6,383,512; 5,783,565; 7,202,227;6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449, all ofwhich are hereby incorporated by reference in their entireties.

In certain embodiments, liposomes used for delivery are amphotericliposomes such SMARTICLES® (Marina Biotech, Inc.) which are described indetail in U.S. Pre-grant Publication No. 20110076322. The surface chargeon the SMARTICLES® is fully reversible which make them particularlysuitable for the delivery of nucleic acids. SMARTICLES® can be deliveredvia injection, remain stable, and aggregate free and cross cellmembranes to deliver the nucleic acids.

Any of the oligonucleotide inhibitors described herein (e.g.,oligonucleotide inhibitors of miR-92a) can be delivered to the targetcell (e.g., a fibrocyte, fibroblast, keratinocyte or endothelial cell)by delivering to the cell an expression vector encoding theoligonucleotide inhibitor. A “vector” is a composition of matter whichcan be used to deliver a nucleic acid of interest to the interior of acell. Numerous vectors are known in the art including, but not limitedto, linear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. Examples ofviral vectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, and the like. In oneparticular embodiment, the viral vector is a lentiviral vector or anadenoviral vector. An expression construct can be replicated in a livingcell, or it can be made synthetically. For purposes of this application,the terms “expression construct,” “expression vector,” and “vector,” areused interchangeably to demonstrate the application of the invention ina general, illustrative sense, and are not intended to limit theinvention.

In one embodiment, an expression vector for expressing anoligonucleotide inhibitor described herein (e.g., oligonucleotideinhibitors of miR-92a) comprises a promoter operably linked to apolynucleotide sequence encoding the oligonucleotide inhibitor. Thephrase “operably linked” or “under transcriptional control” as usedherein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

As used herein, a “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. Suitablepromoters include, but are not limited to RNA pol I, pol II, pol III,and viral promoters (e.g. human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, and the Rous sarcoma virus longterminal repeat). In one embodiment, the promoter is a fibroplastspecific promoter such as the FSP1 promoter, etc. In another embodiment,the promoter is an endothelial specific promoter such as the ICAM-2promoter, etc.

In certain embodiments, the promoter operably linked to a polynucleotideencoding an oligonucleotide inhibitor described herein (e.g.,oligonucleotide inhibitors of miR-92a) can be an inducible promoter.Inducible promoters are known in the art and include, but are notlimited to, tetracycline promoter, metallothionein IIA promoter, heatshock promoter, steroid/thyroid hormone/retinoic acid response elements,the adenovirus late promoter, and the inducible mouse mammary tumorvirus LTR.

Methods of delivering expression constructs and nucleic acids to cellsare known in the art and can include, for example, calcium phosphateco-precipitation, electroporation, microinjection, DEAE-dextran,lipofection, transfection employing polyamine transfection reagents,cell sonication, gene bombardment using high velocity microprojectiles,and receptor-mediated transfection.

One will generally desire to employ appropriate salts and buffers torender delivery vehicles stable and allow for uptake by target cells.Aqueous compositions of the present invention can comprise an effectiveamount of the delivery vehicle comprising the inhibitor polynucleotides(e.g. liposomes or other complexes or expression vectors) dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” includes solvents, buffers, solutions, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like acceptable for use in formulatingpharmaceuticals, such as pharmaceuticals suitable for administration tohumans. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredients of thepresent invention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions, provided they do not inactivate the oligonucleotides ofthe compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal, intraarterial, orintravenous injection. In some embodiments, the pharmaceuticalcomposition is directed injected into lung or cardiac tissue. In someembodiments, the pharmaceutical composition is directly injected intothe wound area. In some embodiments, the pharmaceutical composition istopically applied to the wound area.

Pharmaceutical compositions comprising a miR-92 inhibitor may also beadministered by catheter systems or systems that isolatecoronary/pulmonary circulation for delivering therapeutic agents to theheart and lungs. Various catheter systems for delivering therapeuticagents to the heart and coronary vasculature are known in the art. Somenon-limiting examples of catheter-based delivery methods or coronaryisolation methods suitable for use in the present invention aredisclosed in U.S. Pat. No. 6,416,510; U.S. Pat. No. 6,716,196; U.S. Pat.No. 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent PublicationNo. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S.Patent Publication No. 2007/0060907, which are all herein incorporatedby reference in their entireties. Such compositions would normally beadministered as pharmaceutically acceptable compositions as describedherein.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use, catheter delivery,or inhalational delivery include, for example, sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (e.g. aerosols, nebulizersolutions). Generally, these preparations are sterile and fluid to theextent that easy injectability or aerosolization/nebulization exists.Preparations should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Appropriate solvents ordispersion media may contain, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialan antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In some embodiments, a composition comprising a miR-92 inhibitor issuitable for topical application, such as administration at a woundmargin or wound bed. In some embodiments, the composition compriseswater, saline, PBS or other aqueous solution. In some embodiments, themiR-92 inhibitor is in a lotion, cream, ointment, gel or hydrogel. Insome embodiments, the composition suitable for topical applicationcomprises macromolecule complexes, nanocapsules, microspheres, beads, ora lipid-based system (e.g., oil-in-water emulsions, micelles, mixedmicelles, and liposomes) as a delivery vehicle. In yet anotherembodiment, the miR-92 inhibitor is in the form of a dry powder orincorporated into a wound dressing.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In some embodiments, sterile powders can beadministered directly to the subject (i.e. without reconstitution in adiluent), for example, through an insufflator or inhalation device.

In some embodiments, administration of a miR-92 inhibitor is bysubcutaneous or intradermal injection, such as to a wound (e.g., achronic wound, diabetic foot ulcer, venous stasis leg ulcer or pressuresore). Administration may be at the site of a wound, such as to thewound margin or wound bed.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids), or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like). Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like).

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules, unit dose inhalers, and the like. For parenteraladministration in an aqueous solution, for example, the solutiongenerally is suitably buffered and the liquid diluent first renderedisotonic for example with sufficient saline or glucose. Such aqueoussolutions may be used, for example, for intravenous, intramuscular,subcutaneous, intraarterial, and intraperitoneal administration.Preferably, sterile aqueous media are employed as is known to those ofskill in the art, particularly in light of the present disclosure. Byway of illustration, a single dose may be dissolved in 1 ml of isotonicNaC1 solution and either added to 1000 ml of hypodermoclysis fluid orinjected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, and general safety and puritystandards as required by FDA Office of Biologics standards.

The composition or formulation may employ a plurality of therapeuticoligonucleotides, including at least one described herein. For example,the composition or formulation may employ at least 2, 3, 4, or 5 miR-92inhibitors described herein. In another embodiment, an oligonucleotideof the present invention may be used in combination with othertherapeutic modalities. Combinations may also be achieved by contactinga cell with more than one distinct composition or formulation, at thesame time. Alternatively, combinations may be administered sequentially.

In one embodiment of the present invention, an oligonucleotide inhibitorof miR-92 is used in combination with other therapeutic modalities.Examples of combination therapies include any of the foregoing.Combinations may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, at the same time, wherein onecomposition includes the oligonucleotide inhibitor of miR-92 and onemore other agents. Alternatively, the therapy using an oligonucleotideinhibitor of miR-92 may precede or follow administration of the otheragent(s) by intervals ranging from minutes to weeks. In embodimentswhere the other agent and oligonucleotide inhibitor of miR-92 areapplied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and the oligonucleotide inhibitor ofmiR-92 would still be able to exert an advantageously combined effect onthe cell. In such instances, it is contemplated that one would typicallycontact the cell with both modalities within about 12-24 hours of eachother, within about 6-12 hours of each other, or with a delay time ofonly about 12 hours. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

In one embodiment, more than one administration of the oligonucleotideinhibitor of miR-92 or the other agent(s) will be desired. In thisregard, various combinations may be employed. By way of illustration,where the oligonucleotide inhibitor of miR-92 is “A” and the other agentis “B,” the following permutations based on 3 and 4 totaladministrations are provided as examples: AB/A, B/A/B, B/B/A, A/A/B,B/A/A, A/B/B, B/B/B/A, B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A,B/A/B/A, B/A/A/B, B/B/B/A, A/A/A/B, B/A/A/A, A/B/A/A, A/A/B/A, A/B/B/B,B/A/B/B, B/B/A/B. Other combinations are likewise contemplated. Specificexamples of other agents and therapies are provided below.

In one embodiment of the present invention, the method of promotingangiogenesis in a subject in need thereof comprises administering to thesubject a miR-92 inhibitor, such as an oligonucleotide inhibitor ofmiR-92 as described herein, and another agent that promotesangiogenesis. In one embodiment of the present invention, a method oftreating or preventing peripheral vascular disease (e.g., peripheralartery disease) in a subject in need thereof comprises administering tothe subject a miR-92 inhibitor, such as an oligonucleotide inhibitor ofmiR-92 as described herein. In some embodiments, the method furthercomprises administering another agent with an oligonucleotide inhibitorof miR-92. The other agent may promote angiogenesis or be an agent usedfor treating atherosclerosis or peripheral vascular disease (e.g.,peripheral artery disease). The other agent may be a phosphodiesterasetype 3 inhibitor (such as cilostazol), a statin, an antiplatelet,L-carnitine, propionyl-L-carnitine, pentoxifylline, or naftidrofuryl.The method of treating or preventing peripheral vascular disease (e.g.,peripheral artery disease) in a subject in need thereof may alsocomprise administering oligonucleotide inhibitor of miR-92 to thesubject, in which the subject is also receiving, or will be receivinggene therapy (e.g., with a proangiogenic factor, such as VEGF, FGF,HIF-1α, HGF, or Del-1), cell therapy, and/or antiplatelet therapy. Insome embodiments, the method comprises administering oligonucleotideinhibitor of miR-92 and an antimicrobial to the subject.

In one embodiment of the present invention, the method of promotingwound healing in a subject in need thereof comprises administering tothe subject a miR-92 inhibitor, such as an oligonucleotide inhibitor ofmiR-92 as described herein. In one embodiment, the subject has diabetes.In some embodiments, the subject has a chronic wound, diabetic footulcer, venous stasis leg ulcer or pressure sore. In another embodiment,the subject has peripheral vascular disease (e.g., peripheral arterydisease). In some embodiments, the method further comprisesadministering another agent with an oligonucleotide inhibitor of miR-92.The other agent may be an agent used for treating peripheral vasculardisease (e.g., peripheral artery disease), such as described above. Insome embodiments, the other agent promotes wound healing or is used totreat diabetes. The other agent may be a pro-angiogenic factor. In someembodiments, the other agent is a growth factor, such as VEGF or PDGF.In some embodiments, the other agent promotes VEGF expression oractivity or PDGF expression or activity. In some embodiments, the otheragent is an allogeneic skin substitute or biologic dressing, (e.g.,Dermagraft® or Apligraf®, available from Organogenesis, Canton, Mass.)or a platelet derived growth factor (PDGF) gel, such as becaplermin(Buchberger et al. Experimental and Clinical Endocrinology and Diabetes119:472-479 (2011)). In some embodiments, the other agent is adebridement agent or antimicrobial agent.

The present invention is also based, in part, on the discovery of genessignificantly regulated by miR-92. Accordingly, another aspect of thepresent invention is a method for evaluating or monitoring the efficacyof a therapeutic for modulating angiogenesis or wound healing in asubject receiving the therapeutic comprising: obtaining a sample fromthe subject; measuring the expression of one or more genes listed inTable 3 in the sample; and comparing the expression of the one or moregenes to a pre-determined reference level or level of the one or moregenes in a control sample, wherein the comparison is indicative of theefficacy of the therapeutic. In some embodiments, the therapeuticmodulates miR-92 function and/or activity. The therapeutic can be amiR-92 antagonist, such as a miR-92 oliognucleotide inhibitor selectedfrom Tables 1 and 2. In other embodiments, the therapeutic is a miR-92agonist, such as a miR-92 mimic. In some embodiments, the subjectsuffers from ischemia, myocardial infarction, chronic ischemic heartdisease, peripheral or coronary artery occlusion, ischemic infarction,stroke, atherosclerosis, acute coronary syndrome, coronary arterydisease, carotid artery disease, or peripheral vascular disease (e.g.,peripheral artery disease). In some embodiments, the subject suffersfrom diabetes, a chronic wound, diabetic foot ulcer, venous stasis legulcer or pressure sore.

In some embodiments, the method of evaluating or monitoring the efficacyof a therapeutic for modulating angiogenesis or wound healing in asubject receiving the therapeutic further comprises performing anotherdiagnostic, assay or test evaluating angiogenesis in a subject. In someembodiments, the additional diagnostic assay or test for evaluating ormonitoring the efficacy of a therapeutic for modulating angiogenesis isa walk time test, an ankle-bronchial index (ABI), arteriography orangiography on the subject, or a SPECT analysis.

Another aspect of the present invention is a method for selecting asubject for treatment with a therapeutic that modulates miR-92 functionand/or activity comprising: obtaining a sample from the subject;measuring the expression of one or more genes listed in Table 3 in thesample; and comparing the expression of the one or more genes to apre-determined reference level or level of the one or more genes in acontrol sample, wherein the comparison is indicative of whether thesubject should be selected for treatment with the therapeutic. In someembodiments, the therapeutic is a miR-92 antagonist, such as a miR-92oligonucleotide inhibitor selected from Tables 1 and 2. In otherembodiments, the therapeutic is a miR-92 agonist, such as a miR-92mimic. In some embodiments, the subject suffers from ischemia,myocardial infarction, chronic ischemic heart disease, peripheral orcoronary artery occlusion, ischemic infarction, stroke, atherosclerosis,acute coronary syndrome, coronary artery disease, carotid arterydisease, or peripheral vascular disease (e.g., peripheral arterydisease). In some embodiments, the subject suffers from diabetes, achronic wound, diabetic foot ulcer, venous stasis leg ulcer or pressuresore.

In some embodiments, the method for selecting a subject for treatmentwith a therapeutic that modulates miR-92 function and/or activitycomprises obtaining a sample from a subject treated with thetherapeutic. In some embodiments, the subject is not treated with thetherapeutic and the sample is treated with the therapeutic. In someembodiments, the subject is treated with the therapeutic and the sampleis treated with the therapeutic. In some embodiments, the method furthercomprises performing another diagnostic, assay or test evaluatingangiogenesis or wound healing in a subject. In some embodiments, theadditional diagnostic assay or test for evaluating angiogenesis is awalk time test, an ankle-bronchial index (ABI), arteriography orangiography on the subject, or a SPECT analysis.

The walk test can be a non-invasive treadmill test to measure the changein maximum or pain-free walk time in response to therapy. Theankle-bronchial index (ABI) can be a pressure measurement taken at thearm and the ankle, such as measured by ultrasound. The index can then beexpressed as a ratio of the blood pressure at the ankle compared to thepressure at the arm. The arteriography can be a contrast dye method tomeasure blood flow through arteries or veins. The SPECT (Single PhotonEmission Computed Tomography) analysis can be performed with a 3-Dimaging system using radiation to measure blood flow throughcapillaries.

Also provided herein is a method for evaluating an agent's ability topromote angiogenesis or wound healing comprising: contacting a cell withthe agent; measuring the expression of one or more genes listed in Table3 in the cell contacted with the agent; and comparing the expression ofthe one or more genes to a pre-determined reference level or level ofthe one or more genes in a control sample, wherein the comparison isindicative of the agent's ability to promote angiogenesis. In someembodiments, the method further comprises determining miR-92 functionand/or activity in the cell contacted with the agent. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a cardiac or muscle cell. In some embodiments, the cell is involvedin wound healing. In some embodiments, the cell is a fibrocyte,fibroblast, keratinocyte or endothelial cell. In yet other embodiments,the cell is in vivo or ex vivo.

Measuring or detecting the expression of a gene can be performed in anymanner known to one skilled in the art and such techniques for measuringor detecting the level of a gene are well known and can be readilyemployed. Gene expression levels may be determined measuring the mRNAlevels of a gene or the protein levels of a protein that the geneencodes. A variety of methods for detecting gene expression have beendescribed and include Western blotting, Northern blotting, microarrays,electrochemical methods, bioluminescent, bioluminescent proteinreassembly, BRET-based (BRET: bioluminescence resonance energytransfer), RT-PCR, fluorescence correlation spectroscopy andsurface-enhanced Raman spectroscopy. Commercially available kits canalso be used. The methods for detecting gene expression can includehybridization-based technology platforms and massively-parallel nextgeneration sequencing that allow for detection of multiple genesimultaneously.

In some embodiments, a method for determining the therapeutic efficacyof a therapeutic for treating a condition (e.g., peripheral arterydisease or a wound) in a subject comprises selecting a subject fortreatment with a therapeutic (e.g., a miR-92 oligonucleotide inhibitor),selecting a subject for treatment with a therapeutic (e.g., a miR-92oligonucleotide inhibitor), or evaluating an agent's ability to promoteangiogenesis or wound healing; the level of one or more genes, such asselected from Table 3, is determined.

The gene expression in a sample (e.g. a sample from a subject beingadministered the therapeutic or a sample from a subject or cell culture,in which the sample is treated with the therapeutic), can be compared toa standard amount of the gene present in a sample from a subject withthe condition or in the healthy population, each of which may bereferred to as a reference level. In other embodiments, the level ofgene expression is compared to level in a control sample (a sample notfrom a subject with the condition) or compared to the gene expressionlevel in a sample without treatment, (e.g. taken from a subject prior totreatment with a therapeutic or a sample taken from an untreatedsubject, or a cell culture sample that has not been treated with thetherapeutic). Standard levels for a gene can be determined bydetermining the gene expression level in a sufficiently large number ofsamples obtained from normal, healthy control subjects to obtain apre-determined reference or threshold value. As used herein, “referencevalue” refers to a pre-determined value of the gene expression levelascertained from a known sample.

A standard level can also be determined by determining the geneexpression level in a sample prior to treatment with the therapeutic.Further, standard level information and methods for determining standardlevels can be obtained from publically available databases, as well asother sources. In some embodiments, a known quantity of another genethat is not normally present in the sample is added to the sample (i.e.the sample is spiked with a known quantity of exogenous mRNA or protein)and the level of one or more genes of interest is calculated based onthe known quantity of the spiked mRNA or protein. The comparison of themeasured levels of the one or more genes to a reference amount or thelevel of one or more of the genes in a control sample can be done by anymethod known to a skilled artisan.

According to the present invention, in some embodiments, a difference(increase or decrease) in the measured level of the gene relative to thelevel of the gene in the control sample (e.g., sample in patient priorto treatment or an untreated patient) or a predetermined reference valueis indicative of the therapeutic efficacy of the therapeutic, asubject's selection for treatment with the therapeutic, or an agent'sability to promote or inhibit angiogenesis.

For instance, when the level of one or more genes selected from ACTA2,LACTB, SESN1, and KIAA1598 is decreased when compared to the level in acontrol sample or pre-determined reference value and/or the level of oneor more genes selected from LPCAT4, MYO5A, ERGIC2, LEPREL2, SERPIND1,TSPAN8, ITGA5, NOMA///NOMO2///NOMO3, NPTN, CD93, LOC100507246, MAN2A1,CNEP1R1, EFR3A, UBE2Q2, RNF4, ATP6V1B2, FZD6, MYO1C, PPP3CB, CYYR1,EDEM1, LHFPL2, SEMA3F, UBE2Z is increased in a sample from a subjectbeing administered a therapeutic or a sample is treated with thetherapeutic, the result is indicative of the therapeutic being a miR-92inhibitor and/or promotes angiogenesis, and/or the subject should beselected for treatment with a miR-92 therapeutic (e.g., with a miR-92inhibitor or agonist).

In another example, when the level of one or more genes selected fromACTA2, LACTB, SESN1, and KIAA1598 is increased when compared to thelevel in a control sample or pre-determined reference value and/or thelevel of one or more genes selected from LPCAT4, MYO5A, ERGIC2, LEPREL2,SERPIND1, TSPAN8, ITGA5, NOMA///NOMO2///NOMO3, NPTN, CD93, LOC100507246,MAN2A1, CNEP1R1, EFR3A, UBE2Q2, RNF4, ATP6V1B2, FZD6, MYO1C, PPP3CB,CYYR1, EDEM1, LHFPL2, SEMA3F, UBE2Z is decreased in a sample from asubject being administered a therapeutic or a sample is treated with thetherapeutic, the result is indicative of the therapeutic being a miR-92agonist and/or inhibits angiogenesis, and/or the subject should beselected for treatment with a miR-92 therapeutic (e.g., with a miR-92inhibitor or agonist).

Sampling methods are well known by those skilled in the art and anyapplicable techniques for obtaining biological samples of any type arecontemplated and can be employed with the methods of the presentinvention. (See, e.g., Clinical Proteolytics: Methods and Protocols,Vol. 428 in Methods in Molecular Biology, Ed. Antonia Vlahou (2008),)Samples can include any biological sample from which mRNA or protein canbe isolated. Such samples can include serum, blood, plasma, whole bloodand derivatives thereof, cardiac tissue, muscle, skin, hair, hairfollicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelialtissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid,pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites,cerebrospinal fluid, lymph, cardiac tissue, as well as other tissueextract samples or biopsies, in some embodiments, the biological sampleis plasma or serum.

The biological sample for use in the disclosed methods can be obtainedfrom the subject at any point following the start of the administrationof the therapeutic. In some embodiments, the sample is obtained at least1, 2, 3, or 6 months following the start of the therapeuticintervention. In some embodiments, the sample is obtained least 1, 2, 3,4, 6 or 8 weeks following the start of the therapeutic intervention. Insome embodiments, the sample is obtained at least 1, 2, 3, 4, 5, 6, or 7days following the start of the therapeutic intervention. In someembodiments, the sample is obtained at least 1 hour, 6 hours, 12 hours,18 hours or 24 hours after the start of the therapeutic intervention. Inother embodiments, the sample is obtained at least one week followingthe start of the therapeutic intervention.

The methods of the present invention can also include methods foraltering the treatment regimen of a therapeutic. Altering the treatmentregimen can include but is not limited to changing and/or modifying thetype of therapeutic intervention, the dosage at which the therapeuticintervention is administered, the frequency of administration of thetherapeutic intervention, the route of administration of the therapeuticintervention, as well as any other parameters that would be well knownby a physician to change and/or modify.

In some embodiments, the treatment efficacy can be used to determinewhether to continue a therapeutic intervention. In some embodiments thetreatment efficacy can be used to determine whether to discontinue atherapeutic intervention. In some embodiments the treatment efficacy canbe used to determine whether to modify a therapeutic intervention. Insome embodiments the treatment efficacy can be used to determine whetherto increase or decrease the dosage of a therapeutic intervention. Insome embodiments the treatment efficacy can be used to determine whetherto change the dosing frequency of a therapeutic intervention. In someembodiments, the treatment efficacy can be used to determine whether tochange the number or the frequency of administration of the therapeuticintervention. In some embodiments, the treatment efficacy can be used todetermine whether to change the number of doses per day, per week, timesper day. In some embodiments the treatment efficacy can be used todetermine whether to change the dosage amount.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

EXAMPLES Example 1 Multiple Genes are Significantly Regulated by miR-92aModulation in HUVECs

HUVECs were transfected with miR-92a (1 nM) or oligonucleotide inhibitorof miR-92a (compound A; SEQ ID NO: 7; 10 nM) via lipid-mediatedtransfection, and RNA was isolated 48 hours later for expressionprofiling. Three individual replicates for each treatment were profiled.One sample for miR-92a treatment failed QC and was therefore eliminatedfrom the analysis. The genes were selected on the basis of differentialexpression (FDR p-value<=0.05) for miR-92a treatment versus untreatedcells. Intriguingly, as shown in FIG. 1, this signature of genes isreciprocally regulated by the oligonucleotide inhibitor of miR-92a. Thefact that the entire signature of genes is reciprocally regulated by theantimiR is striking. All of the genes that are down-regulated by miR-92amimic are up-regulated by the oligonucleotide inhibitor of miR-92a, andthe genes up-regulated in response to miR-92a mimic are down-regulatedby the oligonucleotide inhibitor of miR-92a. These genes are listed inTable 3.

TABLE 3 Genes significantly regulated by miR-92a modulation AlternativeName Gene Symbol actin, alpha 2, smooth muscle, aorta ACTA2lysophosphatidylcholine acyltransferase 4 LPCAT4 myosin VA MY05A ERGICand golgi 2 ERGIC2 prolyl 3-hydroxylase 3 LEPREL2 Heparin cofactor IISERPIND1 Tetraspanin 8 TSPAN8 Integrin a5 ITGA5 NODAL modulator 1///NODAL NOMO1///NOMO2///NOMO3 modulator 2/// NODAL modulator 3neuroplastin NPTN CD93 molecule CD93 LOC100507246 mannosidase, alpha,class 2A, member 1 MAN2A1 CTD nuclear envelope phosphatase 1 CNEP1R1regulatory subunit 1 EFR3 homolog A EFR3A ubiquitin conjugating enzymeE2Q family UBE2Q2 member 2 ring finger protein 4 RNF4 ATPase, H+transporting, lysosomal ATP6V1B2 56/58kDa, V1 subunit B2 Frizzledhomolog 6 (Drosophila) FZD6 Myosin VA MY01C lactamase beta LACTB sestrin1 SESN1 protein phosphatase 3, catalytic subunit, PPP3CB beta isozymecysteine/tyrosine-rich 1 CYYR1 ER degradation enhancer, mannosidaseEDEM1 alpha-like 1 lipoma HMGIC fusion partner-like 2 LHFPL2 semadomain, immunoglobulin domain SEMA3F (Ig), short basic domain, secreted,(semaphorin) 3F shootin 1 SHTN1 (KIAA1598) ubiquitin conjugating enzymeE2Z UBE2Z

Genes shown in FIG. 1 and listed in Table 3 were subjected to a PubMedliterature search to identify their potential functions. The genes thathave been reported to have a function in vascular angiogenesis arelisted in Table 4. Table 4 also shows the fold-change of the geneexpression in response to antimiR or miR transfection, as well aswhether or not the 3′UTR of the gene contains a seed sequence targetedby miR-92a.

TABLE 4 Significantly regulated genes have roles in vascularangiogenesis Fold- Fold- Seed- Reported Gene Alternative change changematched angiogenesis symbol name antimiR mimic target function SERPIND1Heparin 1.87 −2.59 promotes cofactor II angiogenesis ITGA5 Integrin a51.38 −2.13

promotes angiogenesis TSPAN8 Tetraspanin 8 1.36 −2.60 promotesangiogenesis, downregulated in PAD samples FZD6 Frizzled 1.15 −1.27

implicated in homolog 6 angiogenesis (Drosophila) CD93 CD93 1.07 −1.28

promotes molecule angiogenesis MYO1C Myosin VA 1.05 −1.33

implicated in angiogenesis

Example 2 Confirmation of Gene Regulation by miR-92a Modulation inHUVECs

As described in Example 1, HUVECs were transfected with miR-92a (5 nM)or oligonucleotide inhibitor of miR-92a (antagomiR; A (SEQ ID NO. 7); B(SEQ ID NO. 63); C (SEQ ID NO. 64); 5 nM each) via lipid-mediatedtransfection. 24 hours after transfection, RNA was isolated regulationof targets identified by microarray profiling in Example 1 was assessedby real time PCR. As shown in FIG. 3, four genes identified bymicroarray profiling are increased in response to miR-92a inhibition anddecreased in response to miR-92a mimic, in an independent HUVEClipid-transfection experiment. The radar plot in FIG. 3 indicates therelative expression of MAN2A1, CNEP1R1, ERGIC2, and CD93 in response tomiR-92a inhibitor or mimic, normalized to HUVECs transfected with lipidwithout oligonucleotide. The thick black line indicates where the geneexpression would be if there were no change, the arrow marks the linethat indicates the gene expression in response to D control oligotransfection.

Example 3 Regulation of Integrin α5 Expression by miR-92a in HUVECs

The regulation of integrin α5 by miR-92a modulation was examined inHUVECs via lipid-mediated transfection (FIG. 2A) or passive delivery(FIG. 2B) of miR-92a (mimic) or antimiR-92a oligonucleotides (antagomiR;A (SEQ ID NO. 7); B (SEQ ID NO. 63); C (SEQ ID NO. 64)) at variousconcentrations. For lipid-mediated transfection, the agents weretransfected at 1 nM, 5, nM, 25 nM, or 50 nM, while for passive deliveryof the oligonucleotides, the cells were exposed to the oligonucleotidesshown in FIG. 2B at a concentration of 0.01 uM, 0.1 uM, or 1 uM. Asshown in FIG. 2A, at 24 hours post-lipid-mediated transfection, therelative expression of integrin α5 mRNA was increased in response tomiR-92a inhibition and decreased in response to miR-92a mimic. Therelative expression of integrin α5 mRNA following lipid-mediatedtransfection was examined by extracting the total RNA from the HUVECcells followed by RT-PCR. The relative expression of integrin α5 mRNA inresponse to miR-92a inhibitors or mimic was normalized to HUVECstransfected with lipid without oligonucleotide. As shown in FIG. 2B, at72 hours post passive delivery, the relative expression of integrin α5was increased vs. the control oligonucleotide at higher concentrationsof oligonucleotides. The relative expression of integrin α5 mRNAfollowing passive delivery was examined by extracting the total RNA fromthe HUVEC cells followed by RT-PCR. The relative expression of integrina5 mRNA in response to miR-92a inhibitors or mimic was normalized toHUVECs transfected without oligonucleotide. In addition to theexamination of the relative mRNA levels, the relative expression of theintergrin α5 protein was examined in the lipid mediated transfectionexperiments. Similar to the mRNA expression results, the relativeexpression of integrin α5 protein 24-hours post lipid-mediatedtransfection also increased following miR-92a inhibition and decreasedin response to miR-92a mimic (see FIG. 2C). The relative expression ofintegrin a5 protein following lipid-mediated transfection was examinedby extracting protein from the HUVEC cells followed by Western Blotanalysis. The relative expression of integrin α5 protein in response tomiR-92a inhibitors or mimic was normalized to untreated HUVECs.

Example 4. Dual luciferase assays for testing of miR-92 inhibitor designactivity.

MiR-92 inhibitors were co-transfected at the indicated concentrationwith a dual-luciferase reporter (FIG. 4). The luciferase reportercontains the binding site to the miR-92a seed sequence in the 3′ UTR ofthe gene, therefore, increased luciferase activity indicates increasedmiR-92a inhibition. Luciferase activity was measured 48 hours aftertransfection. Because all inhibitors showed activity at the 2nM and0.2nM dose, the 0.02nM dose was used to rank-order the inhibitors.Within each inhibitor group in FIG. 4, the 2 nM dose is represented bythe left bar, while the 0.2 nM dose is the middle bar and the 0.02 nMdose is the right bar.

In addition, the effect of the presence of a chemically modifiednitrogenous base, such as for example 5-methylcytosine, on the activityof miR-92 oligonucleotide inhibitors was also tested. To compare theactivity of miR-92 inhibitors with or without 5-methylcytosine, HeLacells were co-transfected with a dual luciferase reporter and either amiR-92a antagomiR, compound A (A; SEQ ID NO. 7), compound B (B; SEQ IDNO. 63), compound B lacking 5-methylcytosine (B minus 5-Me; SEQ ID NO.8), compound C (C; SEQ ID NO. 64), compound C lacking 5-methylcytosine(C minus 5-Me; SEQ ID NO. 9), compound D (D) or a miR-92a mimic as shownin FIG. 9. The HeLa cells were transfected with the compounds at theconcentrations indicated in FIG. 9 (i.e., 10 nM, 1 nM, 0.1 nM, or 0.01nM). As described herein, the luciferase reporter contains the bindingsite to the miR-92a seed sequence in the 3′ UTR of the gene, therefore,increased luciferase activity indicates increased miR-92a inhibition.Normalized luciferase activity was assessed by measurement ofluminescence. Luciferase activity was measured 48 hours aftertransfection. A two-way ANOVA analysis across the entire dose-curve foreach compound revealed that there is a statistically-significantdifference between compound B and B minus 5-Me, where compound B wasmore active than B minus 5-Me (p-value of <0.05 by two-way ANOVA withHom-Sidak multiple comparison post-hoc test). A similar analysis showeda statistically-significant difference between compound C minus5-methylcytosine to compound C was also observed with a p-value of lessthan 0.05 (two-way ANOVA with Hom-Sidak multiple comparison post-hoctest). Compound C was more active than C minus 5-Me.

Example 5 Activity of antimiR-92 Compounds in an In Vivo Model ofImpaired Wound Healing

AntimiR-92 compounds (i.e., miR-92a oligonucleotide inhibitors) weretested in an in vivo chronic wound model for increases in woundangiogenesis, and acceleration of wound healing. Db/db (BKS.Cg Dock(Hom)7 m+/+ Leprdb/j) mice have been shown to develop type II diabetes andwound healing impairments by 6 weeks of age. In two separate studies,age and sex matched adult mice were anesthetized and the dorsum wasdepilated. Two 6 mm diameter excisional punch wounds were made on thebacks of the mice equidistant between shoulders and hips, on either sideof the spine, and the wounds were covered with a semi-occlusivedressing.

Compounds at a dose of 100 nmol (˜0.55mg)/wound were applied at the timeof surgery, and on post-operative days 2, 4 and 8 via intradermalinjection at multiple sites around the wound margin. Recombinant humanVEGF (i.e., rhVEGF-165) at a dose of 1 μg/wound and recombinant PDGF-B(i.e., rhPDGF-B) at a dose of 2 μg/wound were used as positive controlsfor enhanced wound healing. Mice or mice administered a vehicle control(i.e., phosphate buffered saline (PBS))were used as negative controls.

Animals were sacrificed at day 10 post-surgery. Histology analysis wasperformed in order to assess the percentage of reepithelialization, thepercentage of granulation tissue ingrowth, and the thickness andcross-sectional area of neo-epithelium and granulation tissue. Histologyanalysis was performed by fixing one half of each skin wound in 10%neutral buffered formalin for 24 hours and embedding in paraffinaccording to standard protocols. 4 um tissue sections weredeparaffinized and stained with hematoxylin and eosin. Histology imageswere taken under 4-20× magnification and images were analyzed forpercentage reepithelialization, percentage of granulation tissueingrowth, area and thickness and cross-sectional area of neo-epitheliumand granulation tissue using ImageJ (NCBI).

Data from the two studies are presented in FIG. 5A-F and FIG. 6A-F,respectively. FIGS. 5A and 6A illustrate the percentre-epithelialization ((1−[epithelial gap divided by wound width])×100),while FIGS. 5B and 6B show the percent of each wound that was filledwith granulation tissue ((1−[granulation tissue gap divided by woundwidth]×100), FIGS. 5C and 6C show the granulation tissue area within thewound, and FIGS. 5D and 6D show the average thickness of granulationtissue within the wound. VEGF-165 non-significantly increased woundre-epithelialization (FIGS. 5A and 6A) and granulation tissue thickness(FIGS. 5D and 6D), while significantly increasing the percent of eachwound that was filled with granulation tissue (FIGS. 5B and 6B). PDGF-Bsignificantly increased wound re-epithelialization (FIG. 6A) andgranulation tissue ingrowth (FIG. 6B), with a non-significant increasein granulation tissue area (see FIG. 6C) and thickness (FIG. 6D).Conversely, oligonucleotide inhibitors of miR-92 A and C significantlyincreased granulation tissue formation (% granulation tissue filled(FIG. 6B), granulation tissue area (FIGS. 5B and 6B) and averagegranulation tissue thickness (FIGS. 5D and 6D), with A showing someincrease in wound re-epithelialization as well (FIGS. 5A and 6A). Theseresults show that multiple oligonucleotide inhibitors of miR-92accelerated wound healing and new tissue formation to an extent that wasgreater than either VEGF or PDGF peptides.

The mechanism by which oligonucleotide inhibitors of miR-92 accelerateshealing of chronic wounds is believed to be due to increasedangiogenesis. Immunohistochemistry was performed on a subset of groupsfrom the two db/db wound healing studies to assess blood vessel ingrowthand number of endothelial cells, as a measure ofneovascularization/angiogenesis. Immunohistochemistry was performed bystaining 4 μm deparaffinized tissue sections with a primary antibodyspecific for CD31, followed either by fluorescent secondary antibodiesand DAPI to visualize nuclei (first study), or by HRP-conjugatedsecondary antibodies followed by staining with DAB and hematoxylin tovisualize immunocomplexes and nuclei, respectively (second study).Fluorescent images or chromogenic images were taken under 4-20×magnification and the number of CD31+ endothelial cells and bloodvessels were counted at the wound margin using automated thresholdingand image analysis macros in ImageJ. These data are presented in FIG. 5Efor the first study and FIG. 6E for the second study. Similarly, thearea of the tissue that was CD31+ was calculated in an automated fashionusing ImageJ and is presented in FIG. 5F for the first study and FIG. 6Ffor the second study. Both studies demonstrated a significant increasein CD31+ endothelial cells and tissue area that was CD31+ witholigonucleotide inhibitor of miR-92 treatment. PDGF treatment did notaccelerate neoangiogenesis in this study.

The effect of compounds on the mRNA expression of miR-92 target genes(e.g., listed in Table 3) is was measured via quantitative RT-PCR. TotalRNA was isolated from 10-50 mg of skin tissue by homogenizing the tissuewith 1 ml of TRIzol reagent (Life Technologies) in an Omni BeadRuptor.Total RNA isolation was performed per manufacturer's instructions (LifeTechnologies). RNA concentration was measured on a NanoDrop 1000(Thermo). Gene expression was measured with quantitative Real-TimePolymerase Chain Reaction analysis (RT-PCR). For RT-PCR analysis of invivo tissue samples, 100 ng of total RNA was reverse transcribed byMultiScribe RT (Life Technologies) according to the manufacturer'sspecifications. Gene expression was measured with Life TechnologiesTaqman gene expression assays. Gene expression was normalized to ahousekeeping gene such as GAPDH and calculated as relative expressioncompared to the average of the control group. FIG. 7 presents thede-repression of selected miR-92a target genes by oligonucleotideinhibitors of miR-92 from one in vivo study in db/db mouse excisionalwounds.

The effect of compounds on the protein expression of the miR-92 targetITGA5 was evaluated using immunohistochemistry. Immunohistochemistry wasperformed by staining 4 μm deparaffinized tissue sections with a primaryantibody specific for ITGA5, followed by HRP-conjugated secondaryantibodies and then by staining with DAB and hematoxylin to visualizeimmunocomplexes and nuclei, respectively. Chromogenic images were takenunder 4-20× magnification. Representative sections are presented in FIG.8A-D. Little staining was seen in PBS (FIG. 8A) or rhPDGF (FIG. 8B)treated db/db mouse wounds, whereas oligonucleotide inhibitor of miR-92treated (e.g., compound A in FIG. 8C-D) wounds showed high levels ofstaining, localized to endothelial cells and blood vessel walls. Arrowsindicate selected blood vessels in all images; note the lack of stainingin PBS and PDGF groups and the high degree of staining in A treatedwounds. This indicates de-repression of the miR-92a target ITGA5 byantimiR-92a oligonucleotides.

The antimiR-92 compounds enhanced wound healing as compared to thenegative control and as compared to rhVEGF or rhPDGF. The miR-92atargets listed in Table 3 were modulated in the mice administered theoligonucleotide inhibitors of miR-92 that enhance wound healing.

All publications, patents, and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for promoting wound healing in a subject comprisingadministering an oligonucleotide comprising a sequence that is at leastpartially complementary to miR-92, wherein the administration of theoligonucleotide reduces function or activity of miR-92, therebypromoting wound healing.
 2. The method of claim 1, wherein theoligonucleotide comprises at least one locked nucleic acid (LNA)containing a 2′ to 4′ methylene bridge.
 3. The method of claim 1 or 2,wherein the oligonucleotide comprising a sequence that is at leastpartially complementary to miR-92 comprises a sequence of at least 16nucleotides, wherein the sequence comprises no more than threecontiguous LNAs, wherein from the 5′ end to the 3′ end, positions 1, 6,10, 11, 13 and 16 of the sequence are LNAs.
 4. The method of claim 3,wherein from the 5′ end to the 3′ end, the sequence further comprisesLNAs at positions 3, 9, and
 14. 5. The method of claim 3, wherein fromthe 5′ end to the 3′ end, the sequence further comprises LNAs atpositions 3, 8, and
 14. 6. The method of claim 3, wherein from the 5′end to the 3′ end, the sequence further comprises LNAs at positions 5,8, and
 15. 7. The method of any one of claims 4-6, wherein from the 5′end to the 3′ end, the sequence further comprises a deoxyribonucleicacid (DNA) nucleotide at the second nucleotide position.
 8. The methodof claim 7, wherein the DNA nucleotide at the second nucleotide positioncontains a chemically modified nitrogenous base.
 9. The method of claim8, wherein the chemically modified nitrogenous base is 5-methylcytosine.10. The method of any one of claims 1-9, wherein the oligonucleotidecomprises at least one nucleotide that is 2′-deoxy, 2′ O-alkyl or 2′halo modified.
 11. The method of any one of claims 1-10, wherein theoligonucleotide has a 5′ cap structure, 3′ cap structure, or 5′ and 3′cap structure.
 12. The method of any one of claims 1-11, wherein theoligonucleotide comprises one or more phosphorothioate linkages.
 13. Theoligonucleotide of claim 12, wherein the oligonucleotide is fullyphosphorothioate-linked.
 14. The method of any one of claims 1-13,further comprising a pendent lipophilic group.
 15. The method of claim1, wherein the oligonucleotide comprises a sequence selected from Table1 or
 2. 16. The method of any one of claims 1-15, wherein the subject ishuman.
 17. The method of any one of claims 1-16, wherein the subjectsuffers from diabetes.
 18. The method of any one of claims 1-17, whereinthe wound healing is for a chronic wound, diabetic foot ulcer, venousstasis leg ulcer or pressure sore.
 19. The method of any one of claims1-18, wherein the administration of the oligonucleotide produces anincreased rate of re-epithelialization, granulation, and/orneoangiogenesis during wound healing as compared to no treatment ortreatment with an agent known to promote wound healing.
 20. The methodof claim 19, wherein the agent known to promote wound healing isplatelet derived growth factor (PDGF) or vascular endothelial growthfactor (VEGF).
 21. An oligonucleotide comprising a sequence selectedfrom Table
 2. 22. The oligonucleotide of claim 21, wherein at least onenon-locked nucleotide of the oligonucleotide is 2′ deoxy, 2′ O-alkyl or2′ halo modified.
 23. The oligonucleotide of claim 21 or 22, wherein atleast one locked nucleic acid (LNA) of the oligonucleotide has a 2′ to4′ methylene bridge.
 24. The oligonucleotide of any one of claims 21-23,wherein the oligonucleotide has a 5′ cap structure , 3′ cap structure,or 5′ and 3′ cap structure.
 25. The oligonucleotide of any one of claims21-24, wherein the oligonucleotide comprises one or morephosphorothioate linkages.
 26. The oligonucleotide of claim 25, whereinthe oligonucleotide is fully phosphorothioate-linked.
 27. Theoligonucleotide of any one of claims 21-26, further comprising a pendentlipophilic group.
 28. A pharmaceutical composition comprising aneffective amount of the oligonucleotide of any one of claims 21-27, or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent.
 29. The pharmaceuticalcomposition of claim 28, wherein the pharmaceutically-acceptable carriercomprises a colloidal dispersion system, macromolecular complex,nanocapsule, microsphere, bead, oil-in-water emulsion, micelle, mixedmicelle, or liposome.
 30. A method of reducing or inhibiting activity ofmiR-92 in a cell comprising contacting the cell with the oligonucleotideof any one of claims 21-27.
 31. The method of claim 30, wherein the cellis a mammalian cell.
 32. The method of claim 31, wherein the cell is acardiac cell, muscle cell, fibrocyte, fibroblast, keratinocyte orendothelial cell.
 33. The method of any one of claims 30-32, wherein thecell is in vitro, in vivo or ex vivo.
 34. A method of promotingangiogenesis in a subject comprising administering to the subject theoligonucleotide of any one of claims 21-27.
 35. The method of claim 34,wherein the subject suffers from ischemia, myocardial infarction,chronic ischemic heart disease, peripheral or coronary artery occlusion,ischemic infarction, stroke, atherosclerosis, acute coronary syndrome,coronary artery disease, carotid artery disease, diabetes, chronicwound(s), peripheral vascular disease or peripheral artery disease. 36.The method of claim 34 or 35, wherein the subject is a human.
 37. Amethod of treating diabetes, chronic wound(s), ischemia, myocardialinfarction, chronic ischemic heart disease, peripheral or coronaryartery occlusion, ischemic infarction, stroke, atherosclerosis, acutecoronary syndrome, coronary artery disease, carotid artery disease, orperipheral artery disease comprising administering to the subject theoligonucleotide of any one of claims 21-27.
 38. The method of claim 37,wherein the subject is a human.
 39. A method for evaluating ormonitoring the efficacy of a therapeutic for modulating angiogenesis ina subject receiving the therapeutic comprising: a) measuring theexpression of one or more genes listed in Table 3 in a sample from thesubject; and b) comparing the expression of the one or more genes to apre-determined reference level or level of the one or more genes in acontrol sample, wherein the comparison is indicative of the efficacy ofthe therapeutic.
 40. The method of claim 39, further comprisingperforming a walk time test on the subject, determining anankle-bronchial index (ABI) for the subject, performing an arteriographyor angiography on the subject, or performing a SPECT analysis on thesubject.
 41. A method for evaluating or monitoring the efficacy of atherapeutic for modulating wound healing in a subject receiving thetherapeutic comprising: a) measuring the expression of one or more geneslisted in Table 3 in a sample from the subject; and b) comparing theexpression of the one or more genes to a pre-determined reference levelor level of the one or more genes in a control sample, wherein thecomparison is indicative of the efficacy of the therapeutic.
 42. Themethod of any one of claims 39-41, wherein the therapeutic modulatesmiR-92 function and/or activity.
 43. The method of claim 42, wherein thetherapeutic is a miR-92 oligonucleotide inhibitor.
 44. The method ofclaim 43, wherein the miR-92 oligonucleotide inhibitor is selected fromTables 1 and
 2. 45. The method of any one of claims 39-44, wherein thesubject suffers from ischemia, myocardial infarction, chronic ischemicheart disease, peripheral coronary artery occlusion, ischemicinfarction, stroke, atherosclerosis, acute coronary syndrome, coronaryartery disease, carotid artery disease, diabetes, chronic wound(s),peripheral vascular disease or peripheral artery disease.
 46. The methodof any one of claims 39-45, wherein the subject is a human.
 47. A methodfor evaluating an agent's ability to promote angiogenesis or woundhealing comprising: a) measuring the expression of one or more geneslisted in Table 3 in a cell contacted with the agent; and b) comparingthe expression of the one or more genes to a pre-determined referencelevel or level of the one or more genes in a control sample, wherein thecomparison is indicative of the agent's ability to promote angiogenesisor wound healing.
 48. The method of claim 47, further comprisingdetermining miR-92 function and/or activity in the cell contacted withthe agent.
 49. The method of claim 47 or 48, wherein the cell is amammalian cell.
 50. The method of claim 49, wherein the cell is acardiac cell, muscle cell, fibrocyte, fibroblast, keratinocyte orendothelial cell.
 51. The method of any one of claims 47-50, wherein thecell is in vitro, in vivo or ex vivo.
 52. A method for selecting asubject for treatment with a therapeutic that modulates miR-92 functionand/or activity comprising: a) measuring the expression of one or moregenes listed in Table 3 in a sample from the subject, wherein thesubject is treated with the therapeutic; and b) comparing the expressionof the one or more genes to a pre-determined reference level or level ofthe one or more genes in a control sample, wherein the comparison isindicative of whether the subject should be selected for treatment withthe therapeutic.
 53. The method of claim 52, further comprising furthercomprising performing a walk time test on the subject, determining anankle-bronchial index (ABI) for the subject, performing an arteriographyor angiography on the subject, or performing a SPECT analysis on thesubject.
 54. The method of claim 52, further comprising determiningmiR-92 function and/or activity in the sample.
 55. The method of any oneof claim 52-54, wherein the therapeutic is a miR-92 oligonucleotideinhibitor.
 56. The method of claim 55, wherein the miR-92oligonucleotide inhibitor is selected from Tables 1 and
 2. 57. Themethod of any one of claims 52-56, wherein the subject suffers fromischemia, myocardial infarction, chronic ischemic heart disease,peripheral or coronary artery occlusion, ischemic infarction, stroke,atherosclerosis, acute coronary syndrome, coronary artery disease,carotid artery disease, diabetes, chronic wound(s), peripheral vasculardisease or peripheral artery disease.
 58. The method of any one ofclaims 52-57, wherein the subject is a human.